1
Supplementary Information
For
“Neurofilament Subunits are Integral Components of Synapses and Modulate
Neurotransmission and Behavior In Vivo”
Aidong Yuan1,4*, Henry Sershen2,4*, Veeranna1,4*, Balapal S. Basavarajappa
3,6
, Asok
Kumar1,4, Audrey Hashim2, Martin Berg1, Ju-Hyun Lee1, 4, Yutaka Sato1, Mala V. Rao1,4,
Panaiyur S. Mohan1, Victor Dyakin1, Jean-Pierre Julien7, Virginia M-Y Lee8 and Ralph
A. Nixon1,4,5
1
2
Center
for
Dementia
Research,
Neurochemistry
Division,
3
Analytical
Psychopharmacology Division, Nathan Kline Institute, Orangeburg, New York 10962
4
5
Departments of Psychiatry and Cell Biology, New York University School of Medicine,
New York, NY 10016, 6Department of Psychiatry, College of Physicians & Surgeons,
7
Columbia University, New York, NY 10032, Centre de Recherche du Centre Hospitalier
de l'Université Laval, Département d'anatomie et physiologie de l'Université Laval, 2795
boul. Laurier, Québec G1V 4G2, Canada, 8Department of Pathology & Laboratory
Medicine, University of Pennsylvania, Philadelphia, PA 19104
*AY, HS and V contributed equally to this work.
SUPPLEMENTARY MAREIRALS & MATHODS
Generation of transgenic animals
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Adult male or female mice of the C57BL/6J-rich strain, aged 3 - 12 months were used in
all experiments. Both age and gender were matched for a specific experiment. Mice were
housed at 230C on a 12-h light-dark cycle and were maintained on Lab Chow (Purina
Mills, Gray Summit, MO) supplied at libitum. Production of NFL-null (LKO)1, NFMnull (MKO)2, NFH-null (HKO)3 and α-internexin-null (IKO)4 was described before.
NFH and NFL double knockout (HL-DKO) mice were generated by cross-breeding
NFH-null with NFL-null mice. α-internexin, NFH and NFL triple knockout (IHL-TKO)
mice were generated by cross-breeding α-internexin-null, NFH-null with NFL-null mice.
The investigator was blind to the conditions tested as much as possible. The experimental
protocols were approved by NYU/NKI IACUC Committees under the guidelines of the
Institutional Animal Care and Use Committee of the United States.
Drugs and Antibodies
Cocaine HCl and amphetamine sulfate were obtained from Sigma-Aldrich (St. Louis,
MO) and SCH-22390 from Research Biochemicals International (Natick, MA). Mouse
anti-dopamine D1 receptor monoclonal antibody (Catalog number MAB5290) is from
Chemicon International (Temecula, CA). Monoclonal anti-D1 dopamine receptor
antibody (Catalog number D-187) is from Sigma-Aldrich (St. Louis, MO).
Neurofilament antibodies used are mAbs to NF-L (NR4, Catalog number N5139), NF-M
(NN18, Catalog number N5264) and NF-H (N52, Catalog number N0142) (SigmaAldrich, St. Louis, MO), respectively. Neurofilament antibodies also used are mAb to
NFM (RMO-44, Catalog number 13-0500, Invitrogen, Camarillo, CA) and to internexin (Catalog number MAB5224) and polyclonal antibodies to -internexin
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(Catalog number AB5354), NFL (Catalog number AB1983), NFM (Catalog number
AB1987) (Chemicon International, Temecula, CA) and NFH (Catalog number N4142)
(Sigma-Aldrich, St. Louis, MO). Monoclonal anti-phosphorylated NFM RMO55 is from
Dr. Virginia Lee (University of Pennsylvania). Monoclonal anti-phosphorylated NFH
SMI31 (Catalog number SMI-31R) and SMI 34 (Catalog number SMI-34R) antibodies
and anti-nonphosphorylated NFH SMI33 antibody (Catalog number SMI-33R) are from
Biolegend (Dedham, MA). Anti-NFM rabbit polyclonal antibody was also prepared using
purified mouse NFM proteins as antigens. Anti-phospho-p44/42 (Erk1/2) antibody
(Catalog number 9101S) and polyclonal anti-Rho B antibody (Catalog number 2098) are
from Cell Signaling (Danvers, MA). Anti-GAPDH monoclonal antibody (Catalog
number sc-32233) and polyclonal anti-EEA1 antibody (Catalog number sc-33585) are
from Santa Cruz Biotechnology (Dallas, TX). The following antibodies are from SigmaAldrich (St. Louis, MO): monoclonal anti-Na+/K+ ATPase (1 Subunit) antibody
(Catalog number A277); monoclonal anti--tubulin III antibody (T8578); monoclonal
anti-synaptophysin antibody (Catalog number S5768); monoclonal anti-PSD95 antibody
(Catalog number P-246) and polyclonal anti-synapsin I antibody (Catalog number S193).
Analytical methods
Our published methods were used for the following procedures: measurements of
locomotion and stereotypic activity5; tissue preparation, SDS-PAGE, and immunoblot
analysis6; subcellular fractionations of mouse striatum7, 8; immunocytochemical staining9;
immunogold electron microscopy10. Standard procedures11-13 were used to measure longterm potentiation in transverse hippocampal slices (400 m).
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5-trial social memory test
Twenty IHL-TKO mice (10 females and 10 males) were compared with 16 WT control
mice (10 females and 6 males) of the same background strain (C57BL/6-rich). These
mice were 4-6 months old at the time of the test and individually housed for 7-10 days to
permit establishment of a home-cage territory. All test trials were videotaped and
subsequently analyzed for investigation time. The first trial began by introducing a
stimulus female mouse (never-before-met) into the home cage of a mouse for a 1-min
interaction. At the end of the 1-min trial, the stimulus animal was removed and returned
to an individual holding cage. For the second trial, after a 10-min inter-trial interval, the
same stimulus female was introduced to the mouse for 1-min and later removed to the
individual holding cage. For the third and fourth trials, steps were repeated as the second
trial. In the fifth “dishabituation” trial, a different stimulus female mouse was introduced
to the same mouse for 1-min and later removed to the individual holding cage.
Statistical analysis
Sample sizes were chosen according to the standard practice in the field. Significance
was determined using unpaired two-tailed Student’s t test, the Mann-Whitney test or
Two-way ANOVA with Bonferroni’s post hoc test. The variance is similar between the
groups that are statistically compared.
SUPPLEMENTARY RESULTS
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Relative neurofilament subunit levels in MKO and HL-DKO mice
Because NFM deletion influences the levels of other NF subunits 6, we analyzed levels of
NF proteins in striatum, a region critical to the rewarding effect of cocaine, and in cortex
from MKO. As expected, NFM is absent in MKO mice (Supplementary Figure S5a-b).
While NFH levels in MKO mice were unchanged, NFL levels were reduced to 41.5 ±
6.6% and 38.8 ± 15% of normal levels in striatum and cortex, respectively (mean ± SD,
n=4). Complete loss of -internexin, which did not impair D1R responses, had no effect
on NF protein levels 4. Similarly, loss of NFL dramatically lowered NFH but NFM was
still at about 50% of normal levels 14. Finally, in mice null for both NFH and NFL (HLDKO), NFM levels were 48.2 ± 6.1% of normal levels in striatum (mean ± SD, n=4) and
79.3± 9.6% of normal (mean ± SD, n=4) in the cortex (Supplementary Figure S5c-d).
Accordingly, cocaine responses of these mice were not significantly enhanced (p>0.05,
Student’s t- test) compared with those in WT controls, despite a trend toward increased
responsiveness likely reflecting a borderline lowered NFM level in striatum
(Supplementary Figure S5c).
Social interaction deficits in IHL-TKO mice
We conducted 5-trial social memory assay to determine if IHL-TKO mice have social
memory defect. In this test, the subject mouse was given four brief exposures (trials 1-4)
in its home cage to the same stimulus mouse (intruder). In the 5th trial, the subject mouse
encountered an entirely novel stimulus mouse (novel intruder). WT control mice
displayed normal social memory, as demonstrated by a marked habituation (decreased
exploration) during the first 4 trials and a striking dishabituation (increased exploration)
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on the presentation of a novel animal on the 5th trial. In contrast, IHL-TKO mice showed
no significant habituation during the 4 exposures to the stimulus mouse or dishabituation
to the novel stimulus mouse (Supplementary Figure S4). These results indicate that IHLTKO mice exhibit social interaction deficits.
Increased p-ERK in MKO mice after cocaine injection
The higher levels of D1R on plasma membranes of cocaine-sensitized MKO mice were
associated with accentuated cocaine-induced activation of ERKs and increased
phosphorylation of NFs, a major ERK substrate 15 in brain regions (nucleus accumbens,
lateral septal nucleus, and cingulate cortex) linked to cocaine sensitization 16-18. MKO
and WT mice injected subcutaneously with cocaine (25 mg/kg) or 0.9% saline at 20 min,
120 min, or daily for 7 days were given a challenge injection of cocaine 20 min after the
last injection. In saline-treated mice, we could detect phospho-p44/42 ERK1/2
immunoreactivity in neurons within the nucleus accumbens of both WT and MKO mice
although the p-ERK signal was higher in MKO mice (p<0.05, n = 4, Student’s t- test). In
WT mice, p-ERK immunolabeling was rapid but transient; by 120 min after cocaine
administration had returned to almost basal levels. By contrast, in MKO mice, most pERK immunoreactivity in the nucleus accumbens persisted after 120 min. In mice
receiving daily cocaine injections for 7 days, p-ERK immunoreactivity was stronger in
neurons in nucleus accumbens (p<0.05, n = 8, Student’s t-test) and amygdala (p<0.05, n
= 8, Student’s t-test) of MKO mice than in WT controls (Supplementary Figure S6a-c)
and persisted in nucleus accumbens, septal nucleus, caudate, cingulate cortex 19 and
VTA, including the rostral linear nucleus 20.
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Using the same 7 day cocaine injection protocol, we observed that p-ERK colocalized with phosphorylated NF in neuron subpopulations within the nucleus
accumbens and cingulate cortex of WT mice after chronic cocaine administration. In
MKO mice, the numbers of neurons with such co-localization after chronic cocaine
administration were significantly greater (p<0.05, n = 4, Student’s t-test), indicating an
increased level of ERK signaling in these mice (Supplementary Figure S6d-i). Increased
NF phosphorylation was confirmed by Western blot analysis (Supplementary Figure S6jl). Although NFM was absent from MKO mice, NFH phosphorylation identified by
SMI31 or SMI34 antibodies was significantly increased in cocaine-treated MKO
compared with cocaine treated WT controls (p<0.05, Student’s t-test).
NFM modulates D1 receptor- stimulated hippocampal LTP
Dopamine has been shown to induce a long-lasting synaptic potentiation in hippocampal
slices 21, 22 through D1/D5 receptors 23. Consistent with these findings, we found that
application to the bath of 50µM D1/D5 agonist SKF 38393 for 15 min produced long
lasting synaptic potentiation in WT mice. Moreover, this potentiation was enhanced in
MKO mice. Application of SKF 38393 for 15 min produced an enhanced and persisting
increase of the EPSP slopes in NFM null hippocampal slices compared to that in WT
mice (Supplementary Figure S10). The synaptic potentiation induced by the D1/D5
agonist SKF 38393 had a very slow onset. Potentiation started 40-50 min after
application and the mean potentiation 2 hr after application of SKF 38393 was 171.17 %
± 4.8 % (n=6) for WT mice and 198 ± 5.5 (Mean ± SEM, n=6) for MKO mice (p <0.01,
Student’s t- test). This action of SKF 38393 was specific for D1/D5 receptors: a specific
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antagonist of the D1/D5 receptors, SCH 23390, prevented the potentiation in
hippocampal slices from both WT and MKO mice (Supplementary Figure S10).
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SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure S1: Absence of significant degradation products of NFM from
synaptic NF preparations of mouse striatum by antibody to NFM. Lanes 1-3 were from
three different WT mice and lanes 4-6 from three different MKO animals.
Supplementary Figure S2: Morphometric confirmation of post-embedding
immunogold procedure for localization of NF proteins within synapses. An alternative
pre-embedding method using positive (a; b is an inset) (myelinated axon, linear labeling
of NF with anti-NFM) and negative controls (c, d) (IHL-TKO mice, no labeling with
NFM) confirms NF protein localization within synapses. (e) Synaptic labeling with
monoclonal anti-NFM. (f) Synaptic labeling with polyclonal anti-NFL. (g) Synaptic
labeling with polyclonal anti-NFH. (h) Synaptic labeling with monoclonal anti-internexin. (i) Morphometry of NF-gold particles shows much greater immunogold
labeling in wild-type mice (p<0.0001) per unit area than in IHL-TKO mice. Scale bars,
250nm in a, c, 80nm in b, 400nm in d, 200nm in e, f, 150nm in g, h.
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Supplementary Figure S3: EM images of isolated striatal synaptosomes (a and b).
Mito: mitochondrion; SV: synaptic vesicles; PSD: postsynaptic density. Scale bar: 100nm
in a and b.
Supplementary Figure S4: Social interaction deficits in IHL-TKO mice based on the 5trial social memory test. WT mice displaying normal social recognition showed the
standard pattern of high socialization time (trial 1) followed by decreased socialization
time (trials 2-4; P = 0.001 at trial 3 and P = 0.0005 at trial 4) and increased socialization
time with a novel mouse (trial 5, P < 0.0001). IHL-TKO mice displaying social
recognition defects showed a disrupted pattern of habituation to the same mouse (trials 14) and dishabituation to a novel mouse (trial 5, P = 0.9841).
Supplementary Figure S5: Immunoblot analyses of relative neurofilament subunit levels
in wild-type, MKO and HL-DKO mice. Relative levels of each subunit protein were
normalized to total protein and quantified by using mAbs to NFH, NFM and NFL and
NIH imaging software. As expected, NFM was absent in MKO (a, b) and NFH and NFL
were absent in HL-DKO mice (c, d). In the absence of NFM, NFH slightly increased in
striatum (P = 0.0571) and frontal cortex (P = 0.0235) while NFL significantly decreased
in both striatum (P = 0.0006) and frontal cortex (P = 0.029). In the absence of NFH and
NFL, NFM significantly decreased in striatum (P = 0.0001) and slightly decreased in
frontal cortex (P = 0.0882). Each measurement is the mean ± SD from 4 animals. HL:
HL-DKO.
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Supplementary Figure S6: Increased p-ERK in MKO mice after cocaine injection.
Increased p-ERK in neurons of nucleus accumbens (NA) and amygdala of MKO mice
compared with wild-type 120 min after cocaine injection (a-c). Increased p-Erk
colocalized with phosphorylated NFs in neurons of cingulate cortex following
subcutaneous injections of cocaine (25mg/kg) and analyzed after 7 days (20 min after the
last injection) (d-i). Increased phosphorylated NFH in neurons of striatum, identified by
SMI31 (k, Mean ± SD, n=3, p=0.0369) or SMI34 (l, Mean± SD, n=3, p=0.0418), were
also demonstrated by western blotting in cocaine treated MKO mice compared with
cocaine treated wild-type mice (j). sal: saline; coc: cocaine; β3Tub: β3 tubulin. p-ERK:
phosphorylated ERK.
Supplementary Figure S7: Levels of dopamine, serotonin, and their metabolites in the
absence of NFM. Levels of dopamine, serotonin, and their metabolites in striatum (a),
hippocampus (b) and frontal cortex (v), measured using HPLC-electrochemical detection
methods revealed no major differences in biogenic amine levels in MKO mice (black bar)
compared with wild-type controls (white bar) (P>0.05 for all the metabolites in 3
different areas). Measurements are mean ± SEM from at least 8 animals for each group.
Supplementary Figure S8: The total level of D1 receptor is not altered significantly in
the striatum of MKO mice (p = 0.62 Student’s t- text, n=4) (a, b).
Supplementary Figure S9: Enrichment of plasma membrane and endosome fractions.
Plasma membrane-enriched and endosome-enriched fractions of mouse striata were
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isolated following subcutaneous injections of cocaine (25mg/kg/day) or saline for 7 days.
The enrichment was confirmed by plasma membrane marker ATPase or endosome
marker EEA1.
Supplementary Figure S10: Enhanced D1/D5 receptor agonist –induced long lasting
synaptic potentiation in the absence of NFM. (a) SKF 38393 (50µM; 15 min) applied into
bath solution (bar) induced an increase in the slope of EPSP in both wild-type and MKO
mice hippocampal slices (mean ± SEM; n=6). Combined application of D1/D5 receptor
antagonist SCH 23390 (2µM; 30 min) with SKF 38393 prevented the SKF-induced
potentiation in both wild type and NFM null mice hippocampal slices (n = 6). b:
Representative EPSP traces obtained before and 2hr after the application of SKF 3893.