Supplementary Information
Imbalance of neurotrophin receptor isoforms TrkB-FL/TrkB-T1 induces
neuronal death in excitotoxicity
Óscar G. Vidaurre*, Sergio Gascón*, Rubén Deogracias, Mónica Sobrado, Eloy
Cuadrado, Joan Montaner, Ángeles Rodríguez-Peña, and Margarita Díaz-Guerra
Supplementary Materials and Methods
Chemicals
NMDAR antagonists 2-amino-5-phosphonopentanoic acid (DL-AP5) and ifenprodil
were obtained from Tocris-Cookson (Bristol, UK), and ketamine and memantine were from
Ascent Scientific (North Somerset, UK). N-Methyl-D-Aspartate (NMDA), glutamate,
glycine, cytosine -D-arabinofuranoside (AraC), poly-L-lysine, L-laminin, ionophore
A23187 and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) were from Sigma
Co (St. Louis, Mo, USA). Carbobenzoxy-valinyl-phenylalaninal (calpain inhibitor III, herein
CiIII), lactacystin, calpeptin and calpain I (-calpain) were from Calbiochem (Darmstadt,
Germany). Thapsigargin was obtained from Alomone Labs (Jerusalem, Israel). BCA reagent
was from Pierce (Thermo Scientific, Rockford, IL, USA), and purified BDNF was from
Regeneron Pharmaceuticals (Tarrytown, NY, USA).
Generation of the LV-TrkB-T1/GFP plasmid
The cDNA for rat TrkB-T1 was amplified by PCR from vector pFRK42, provided by
M.
Barbacid
(CNIO,
Madrid,
Spain),
using
oligonucleotides
5’-
AAGGATCCCGTGATGTTCCAGCCAC-3’
and
5’-
AACTCGAGTAAACAGAAGCAATTAATGTCAG-3’. The BamHI and XhoI sites are
underlined, and the TrkB-T1 sequences are in italics. The PCR product digested with these
enzymes was introduced into LV-DsRed/GFP (Gascon et al, 2008a) digested similarly,
substituting the DsRed coding sequence. In this way, we produced the dual lentiviral vector
LV-TrkB-T1/GFP where the synthesis of TrkB-T1 and GFP genes was under the control of
neuro-specific synapsin promoters (pSyn) and their sequences were followed by woodchuck
hepatitis post-transcriptional regulatory elements (WPRE) for the enhancement of
expression.
Primary antibodies
We used polyclonal antibodies specific for the TrkB extracellular domain (panTrkB,
sc-8316), the C-terminal cytoplasmic region of TrkB-FL (sc-12) or the short intracellular
sequence of TrkB-T1 (sc-119), all of them from Santa Cruz Biotechnology (Santa Cruz, CA,
USA), phospho-TrkA (Tyr490; #9141, Cell Signalling, Beverly, MA, USA), neuron-specific
enolase (NSE; AB951, Chemicon, Temacula, CA, USA), grp78/BiP (ADI-SPA-826-D,
Enzo Life Sciences, Plymouth, PA, USA) and GFP (A6455, Invitrogen, Carlsbad, CA,
USA). We also employed monoclonal antibodies for III-tubulin (MAB1637) and nonerythroid spectrin (MAB1622) from Chemicon (Temacula, CA, USA), Neuronal Nuclei
protein (NeuN) (AB13938, Abcam, Cambridge, UK), HA (MMS-101R, Covance Research
Products, Emeryville, CA, USA), -actin (#5441, Sigma Co. St. Louis, Mo, USA), GFP
(#11814460, Roche Diagnostics, Indianapolis, IN, USA) and GAPDH (AM4300,
Ambion/Applied Biosystems, Austin, TX, USA).
Secondary antibodies
For immunoblot, we used goat secondary antibodies coupled to horseradish peroxidase
from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Immunohistochemistry of human
necropsies was performed with secondary antibodies Alexa-Fluor 488-conjugated mousespecific or 568-conjugated rabbit-specific IgG (Invitrogen, Carlsbad, CA, USA). In the case
of the MCAO model, we used Alexa-Fluor 647-conjugated mouse-specific and 488- or 555conjugated rabbit-specific IgG (Molecular Probes, Eugene, OR, USA).
Assessment of neuronal injury in primary cultures treated with NMDA
Cell viability of cortical cultures treated with 100 M NMDA and 10 M glycine was
measured using the MTT reduction assay, as described previously (Gascon et al, 2008b).
The contribution of glial cells in the mixed cultures to absorbance was established by
exposing sister cultures to 400 M NMDA and 10 M glycine for 24 h, conditions which
induce near complete neuronal death and no glial damage. This value was then subtracted
from all of the absorbance measurements to establish the specific neuronal viability. Results
are represented relative to the untreated cells, which were arbitrarily given a 100% value. In
the case of infected cultures, results are relative to cells infected with the same multiplicity
of a control virus not treated with NMDA.
Lentivirus production and titration
Lentiviruses were produced as described previously (Gascon et al, 2008b). For
concentration, the viral suspension was first filtered using a Steriflip-HV 0.45-m filter unit
(Millipore, Billerica, MA, USA) and ultracentrifuged at 20,000 rpm for 2 h in a SW28
Beckman Coulter rotor. Viral pellets were resuspended overnight at 4ºC in PBS. Titers were
established in PC12 cells using FACScan analysis of GFP expression (Gascon et al, 2008b).
In experiments using the syn-HA-TrkB-FL-syn-2xmiT1 virus, which did not encode
fluorescent proteins, we used RNA titration for viral titration. Briefly, viral RNAs were
extracted from 5 l of concentrated viral suspension using an RNeasy micro kit (Qiagen,
Germantown, MD, USA) followed by the removal of potentially contaminating plasmid
DNAs by DNase treatment. A fixed volume of the RNA preparation was reverse-transcribed
to prepare the cDNA, and quantitative PCR was performed using specific primers (5’ACTTGAAAGCGAAAGGGAAAC-3’ and 5’- CACCCATCTCTCTCCTTCTAGCC-3’)
and a Taqman probe (5’-FAM-AGCTCTCTCGACGCAGGACTCGGC-MGB-3’) directed
against the packaging region in the viral genome. Lentiviral RNA titers, expressed as
particles ml-1, were calculated from a standard curve obtained by the serial dilution of a
plasmid that contained the lentiviral packaging region.
Quantitative RT-PCR
Total RNA was reverse-transcribed by random primer extension using a High
Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA).
PCR reactions contained Power SYBR Green PCR Master Mix (Applied Biosystems, Foster
City, CA, USA) with cDNA (5 ng) and specific oligonucleotides (300 nM) and were
performed using a 7900HT Fast Real Time PCR thermocycler (Applied Biosystems, Foster
City, CA, USA). Denaturation at 95ºC for 10 min was followed by 40 cycles of 95°C for 15
s and 60°C for 1 min. The oligonucleotides were: 5’-CCAGTAAGTGCGGGTCATAAGC
and
5’-CCTCACTAAACCATCCAATCGG
for
18S
rRNA;
5’-
GGGGCTGTGCTGCTTGGT and 5’-GCTGCGGACATCTTTGGAGA for TrkB-T1; and
5’-ATCTTCACCCACCTCAAACC and 5’-GAAACCATTCTCCCCGAAAC for TrkB-FL.
Levels of TrkB transcripts were normalized to the levels of 18S rRNA present in the same
samples and are represented as values relative to the levels obtained in the untreated cells.
Supplementary Figure Legends
Supplementary Figure 1 Transient ischemia followed by brief reperfusion induces mild
infarcts in adult rat brain. Nissl staining of coronal sections of animals subjected to 1 h
MCAO followed by 6 h of blood reperfusion showed a hypochromatic area in the ipsilateral
neocortex (A). Detailed comparison of equivalent regions of the ipsilateral (B) and
contralateral hemispheres (C) revealed a loss of cytoplasmic Nissl staining throughout the
lesion, nuclear condensation and characteristic changes in the morphology and size of cell
bodies and processes. All these changes are indicative of progressive neuronal loss in the
infarcted area that results from ischemic damage.
Supplementary Figure S2 Transient cerebral ischemia induces a switch in TrkB expression
in neurons. Double immunohistochemistry of coronal sections of animals subjected to
MCAO followed by 6 h of reperfusion demonstrated an increase in the expression of TrkBT1 in the ischemic region (A) compared to the contralateral hemisphere (B) in cells that
were
not
stained
with
a
GFAP
antibody
specific
for
glial
cells
(arrows).
Immunohistochemistry with panTrkB antibody, which recognizes all of the TrkB isoforms,
probed TrkB expression in cells in both ischemic (C) and contralateral regions (D).
According to previous results, TrkB-FL is the main isoform in the contralateral area, but
TrkB-T1 is predominant in the infarcted region. Representative confocal microscopy images
corresponding to single sections are shown. The scale bar represents 20 m.
Supplementary Figure S3 Treatment of primary cortical cultures with NMDA induces a
progressive decrease of neuronal viability as measured by the MTT assay. The contribution
of glial cells to cell viability in these mixed cultures was established as described in
Supplementary Material and Methods. The relative neuronal viability (mean ± S.E.M., n =
6) is expressed as the percentage of the value obtained in untreated cells (arbitrarily
established as 100%). Student’s unpaired t test was used to assess differences between
treated and untreated cells (**P < 0.01, ***P < 0.001).
Supplementary Figure S4 Regulation of TrkB requires activation of GluN2B-containing
NMDARs. (A) The dysegulation of TrkB induced by NMDA treatment (100 M) was
prevented by the GluN2B-selective antagonist ifenprodil (10 M) similarly to the generic
NMDAR antagonist DL-AP5 (200 M), demonstrating that GluN2B-containing NMDARs
are triggering this regulation. (B) Relative levels of TrkB-FL (mean ± S.E.M., n = 3
experiments) were established after normalization to NSE and comparison to levels in
untreated cells (arbitrarily given a 100% value). Student’s unpaired t test was used to assess
differences between treated and untreated cells (**P < 0.01). (C) Treatment of primary
cultures with thapsigargin (2 M) or the ionophore A23187 (1 M) for 3 h could not mimic
the TrkB isoform imbalance induced by NMDA although expression of the endoplasmic
reticulum stress-responsive protein grp78/BiP was stimulated, reflecting a modification in
calcium homeostasis.
Supplementary Figure S5 Regulation of TrkB and neuronal death requires NMDAR
activation with NMDA concentrations which depend on the cellular composition of the
culture. (A) Analysis of cultures stimulated with glycine (10 M) and different
concentrations of NMDA (1 to 100 M) for 6 h demonstrated dramatic TrkB dysregulation
for NMDA concentrations > 10 M in mixed cultures of neurons and glial cells grown for
14 DIVs in MEM or neuronal-enriched cultures obtained in NB medium. (B) Detailed
comparison of the effect on TrkB-FL regulation of treatment of cultures of distinct cellular
composition with different NMDA concentrations. The relative TrkB-FL levels (mean ±
S.E.M., n = 3 experiments) found in cultures treated with NMDA as before were compared
with those obtained in cultures grown in the same medium with no agonist, arbitrarily
assigned a 100% value. Student’s unpaired t test was used to assess differences between
TrkB-FL levels in neurons grown in MEM or NB and treated with the same NMDA
concentration. The only significant difference was found for a NMDA concentration of 10
µM (P= 0.04). (C) Effect on neuronal viability of NMDA treatment showing an increased
sensitivity of neuronal-enriched cultures over mixed cultures. The relative viability of
neurons grown in MEM or NB medium treated with increasing concentrations of NMDA for
6 h was calculated as before. The results (mean ± S.E.M., n = 3) were expressed as the
percentage of the value obtained in untreated cells grown in the same medium (arbitrarily
established as 100%). Student’s unpaired t test was used to assess differences between
cultures grown in different medium but treated with the same concentration of NMDA (N.S.
nonsignificant, **P < 0.01).
Supplementary Figure S6 Oligonucleotides designed for knock-down of rat TrkB-T1
expression. Top and bottom sequences selected to produce double-stranded oligonucleotides
encoding pre-miRNAs specific for sequences in the 3’-UTR of TrkB-T1 mRNA (miT1) or
unrelated sequences (miC) are shown. These sequences were cloned into the pcDNA6.2GW/miRNA vector to produce pcD/miC and five different pcD/miT1 plasmids. The
portions of these oligonucleotides that are homologous to TrkB-T1 are in bolded italic
characters.
Supplementary Figure S7 TrkB-T1 miRNA-targeted sequences and initial analysis of
TrkB-T1 pre-miRNAs efficiency in an heterologous system. (A) A fragment of TrkB-T1 3’UTR is shown with the target sequences that were included in the various pcD/miT1
plasmids highlighted in the light gray boxes. A variant of the pcD/miT1(2214) plasmid,
pcD/miT1(2210), contains a longer TrkB-T1 nucleotide sequence that is marked in dark
gray. (B) HEK293 cell cultures, which do not express TrkB-T1, were co-transfected with
plasmid LV-TrkB-T1/GFP to drive TrkB-T1 expression from a CMV promoter, which is
upstream of the LTR that is lost in the viral genome, and reporter plasmid pEGFP1-C1
together with different pcD/mT1 plasmids. The latter encode pre-miRNAs specific for the
indicated TrkB-T1 sequences or the negative control pcD/miC, which encodes an unrelated
sequence. Immunoblot analysis of cells transfected for 24 h with TrkB-T1, GFP or -actin
antibodies revealed specific and efficient interference of TrkB-T1 expression induced by the
pcD/mT1(2178) and (2310) plasmids. These plasmids were chosen for further analysis.
Supplementary Figure S8 Basal viability of cortical neurons is not modified by increased
levels of TrkB-FL and/or interference of TrkB-T1 expression. Relative neuronal viability
(mean ± S.E.M., n = 3) of cultures transduced with the same multiplicity of lentiviruses LVHA-TrkB-FL/2xmiT1, LV-HA-TrkB-FL/GFP and LV-miT1/GFP, capable to modify the
TrkB-FL/TrkB-T1 balance, or control viruses LV-DsRed/GFP and LV-miC/GFP (m.o.i. =
2). Results are expressed as percentages of the values obtained in cells infected with LVDsRed/GFP (arbitrarily established as 100%). Student’s unpaired t test demonstrated that the
observed differences were not statistically significant.