Recombinant human TNF-binding protein-1 (rhTBP-1) treatment delays both symptoms progression and motor neuron loss in the wobbler mouse.
ABSTRACT TNF-alpha overexpression may contribute to motor neuron death in amyotrophic lateral sclerosis (ALS). We investigated the intracellular pathway associated with TNF-alpha in the wobbler mouse, a murine model of ALS, at the onset of symptoms. TNF-alpha and TNFR1 overexpression and JNK/p38MAPK phosphorylation occurred in neurons and microglia in early symptomatic mice, suggesting that this activation may contribute to motor neuron damage. The involvement of TNF-alpha was further confirmed by the protective effect of treatment with rhTNF-alpha binding protein (rhTBP-1) from 4 to 9 weeks of age. rhTBP-1 reduced the progression of symptoms, motor neuron loss, gliosis and JNK/p38MAPK phosphorylation in wobbler mice, but did not reduce TNF-alpha and TNFR1 levels. rhTBP-1 might possibly bind TNF-alpha and reduce the downstream phosphorylation of two main effectors of the neuroinflammatory response, p38MAPK and JNK.
- Nature Genetics - NAT GENET. 01/1994; 7(3):425-428.
- [show abstract] [hide abstract]
ABSTRACT: Mice homozygous for the spontaneous motor neuron degeneration mutation (mnd) show at the age of 8 months a marked impairment of the motor function and accumulation of lipofuscin granules in the cytoplasm of almost all neurons of the central nervous system. We previously reported a significant increase in GFAP protein levels in the lumbar spinal cord homogenates by western blot analysis and upregulation of TNF, a proinflammatory cytokine, in the motor neurons of lumbar spinal cord of mnd mice, already in a presymptomatic stage (4 months of age). In the present study, using immunohistochemical analysis, we performed a time course in mnd mice (1, 4 and 9 months of age) evaluating the expression and the distribution of astroglial and microglial cells and the expression of both TNF receptors, TNFR-I and TNFR-II. We observed a marked increase in astroglial and microglial cells and in TNFR-I immunoreactivity already at the 4th month. Since motor neuron dysfunction occurs in mnd mice in the absence of evident loss of spinal motor neurons, the present results indicate that the activation of microglial cells and astrocytes is independent from neuronal degeneration. The role of TNF and TNFR-I on motor neurons is still to be demonstrated.Cytokine 03/2004; 25(3):127-35. · 2.52 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The effect of tumor necrosis factor binding protein (TNFbp) was studied in mice subjected to a permanent middle cerebral artery occlusion (MCAO). TNFbp is a dimeric form of the type I soluble TNF receptor linked to polyethylene glycol (TNFbp), and binds and inhibits TNF-alpha. TNFbp produced a significant reduction in the cortical infarct volume (22.6 +/- 3.5 mm3 immediately after MCAO; 25.2 +/- 2.4 mm3 1 h after MCAO) compared with vehicle-treated animals (30.3 +/- 3.7 mm3 immediately post MCAO; 31 +/- 3.7 mm3 1 h after MCAO (mean +/- S.D.) when administered intracranially up to 60 min post-occlusion. The neuroprotective effect of TNFbp was sustained in mice for 2 weeks after MCAO. DNA fragmentation at the margin of the cortical infarcts was dramatically reduced in mice treated with TNFbp whereas all control animals showed consistent and obvious DNA fragmentation 2 weeks after MCAO. TNFbp could have therapeutic value for the treatment of ischemic stroke if the problem of delivery to brain can be overcome.Brain Research 01/1998; 778(2):265-71. · 2.88 Impact Factor
Recombinant human TNF-binding protein-1 (rhTBP-1) treatment
delays both symptoms progression and motor neuron loss in the
Paolo Bigini,a,⁎Mariaelena Repici,bGiuseppina Cantarella,cElena Fumagalli,aSara Barbera,a
Alfredo Cagnotto,aAda De Luigi,aRossella Tonelli,aRenato Bernardini,c
Tiziana Borsello,b,dand Tiziana Menninia
aDepartment of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa, 19, 20156 Milano, Italy
bDBCM, University of Lausanne, Rue du Bugnon 9, CH-1005 Lausanne, Switzerland
cDepartment of Experimental and Clinical Pharmacology, University of Catania, Viale Andrea Doria, 6, I-95125 Catania, Italy
dDepartment of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa, 19, 20156 Milano, Italy
Received 15 June 2007; revised 16 October 2007; accepted 5 November 2007
Available online 12 November 2007
TNF-α overexpression may contribute to motor neuron death in
amyotrophic lateral sclerosis (ALS). We investigated the intracellular
pathway associated with TNF-α in the wobbler mouse, a murine model
of ALS, at the onset of symptoms. TNF-α and TNFR1 overexpression
and JNK/p38MAPK phosphorylation occurred in neurons and micro-
glia in early symptomatic mice, suggesting that this activation may
contribute to motor neuron damage. The involvement of TNF-α was
further confirmed by the protective effect of treatment with rhTNF-α
binding protein (rhTBP-1) from 4 to 9 weeks of age. rhTBP-1 reduced
the progression of symptoms, motor neuron loss, gliosis and JNK/
downstream phosphorylation of two main effectors of the neuroin-
flammatory response, p38MAPK and JNK.
© 2007 Elsevier Inc. All rights reserved.
Wobbler; Amyotrophic lateral sclerosis; TNF-α; Glial activation
Amyotrophic lateral sclerosis (ALS) is a progressive neurode-
generative disorder primarily affecting motor neurons and leading
to denervation, muscular atrophy and paralysis with a poor prog-
nosis (average survival from symptom onset is b5 years). No
treatments are available. Except in a small percentage of cases
involving identified gene mutations (Hentati et al., 1994; Rosen,
1993), the causes of motor neuron death in ALS are still not
Glial-induced neuroinflammation been proposed as one of
the potential etiological factors (Beghi and Mennini, 2004;
Ghezzi and Mennini, 2001). Although TNF-α levels were high
in ALS patients (Poloni et al., 2000), its role in motor neuron
degeneration is not clear (Ghezzi and Mennini, 2001). This
might be related to the extreme complexity of its intracellular
pathways and the different expression of TNFR1 and TNFR2 in
different CNS regions. In fact, besides its toxic effect, TNF-α
might induce the expression of neurotrophic and anti-inflamma-
tory factors, like IL-6 and LIF (Benigni et al., 1996; Ding et al.,
1995), two cytokines that slow motor neuron disease progres-
sion in the wobbler mouse, a murine model of ALS (Ikeda et
al., 1995; Kurek et al., 1998). The wobbler mouse, carrying a
mutation in the Vps54 gene (Schmitt-John et al., 2005) coding
for a protein involved in the retrograde transport of late
endosomes from the periphery to the Golgi apparatus (Liewen et
al., 2005), shows early-onset selective motor neuron death in the
cervical spinal cord (reviewed in Beghi and Mennini, 2004).
Glial activation (Bigini et al., 2001; Boillee et al., 2001; Rathke-
Hartlieb et al., 1999) and upregulation of TNF-α (Schlomann et
al., 2000) have been reported in the cervical spinal cord of
presymptomatic or early symptomatic mice.
To verify the link between glial activation and TNF-α-induced
neuroinflammation, we evaluated TNF-α and TNFR expression in
the cervical spinal cord region of early symptomatic wobbler mice.
We also investigated the involvement of stress kinases associated
with TNFR activation by measuring the rate of phosphorylation of
JNK and p38MAPK by Western blot and immunohistochemistry.
Finally, to investigate the role of TNF-α in motor neuron de-
generation, we designed experiments in wobbler mice using a TNF-
α binding protein (rhTBP-1). This strategy was aimed at buffering
Neurobiology of Disease 29 (2008) 465–476
⁎Corresponding author. Fax: +39 0239014744.
E-mail address: email@example.com (P. Bigini).
Available online on ScienceDirect (www.sciencedirect.com).
0969-9961/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
of the recombinant human soluble TNFR1 binding protein linked to
polyethylene glycol, acting as a TNF-α inhibitor. rhTBP-1 was
neuroprotective in the rat head injury model and in mouse focal
cerebral ischemia (Nawashiro et al., 1997a,b; Shohami et al., 1996).
In wobbler mice, rhTBP-1 treatment started from the onset of
Materials and methods
Procedures involving animals and their care were conducted in
conformity with the institutional guidelines that are in compliance
with national (D.L. no. 116, G.U. suppl. 40, 18 Febbraio 1992,
circolare no. 8, G.U. 14 Luglio 1994) and international laws and
policies (EEC Council Directive 86/609, OJ L 358, 1, December
12, 1987; NIH Guide for the Care and Use of Laboratory Animals,
US National Research Council 1996).
Homozygous wobbler mice and healthy littermates (NFR/wr
strain, NIH Animal Resources, Bethesda, USA) were bred at
Charles River Italia (Calco, Lecco, Italy). Mice were maintained
at 21±1 °C with relative humidity 55±10% and a 12-h light/
dark cycle. Food (standard pellets) and water were supplied ad
rhTBP-1 treatment protocol
rhTBP-1 (Onercept) was kindly provided by Serono (Ardea,
Rome, Italy). After clear diagnosis of disease at 3 weeks of age
based on phenotype analysis, a total number of 16 wobbler mice
and the same number of healthy littermates (controls) were ran-
domly enrolled to treatment. The experimental groups (8 mice per
group) were: vehicle-treated control and vehicle-treated wobbler
mice, rhTBP-1-treated control and rhTBP-1-treated wobbler mice.
All experimental groups included the same numbers of male and
female mice, since no sex-related differences have been detected in
the course of the wobbler disease.
Mice were injected subcutaneously three times a week with
rhTBP-1 at the dose of 10 mg/kg of body weight (final concen-
tration 2 mg/ml diluted in 40 mM sodium phosphate, 10 mM NaCl,
pH 7) or with the same volume of vehicle (40 mM sodium phos-
phate, 10 mM NaCl, pH 7); treatment continued for 5 weeks, from
4 to the 9 weeks of age. This treatment schedule was selected based
on unreported data from Serono, indicating that in the 3 30 mg/kg
range, rhTBP-1 had optimal anti-inflammatory activity in mice and
was well tolerated for chronic treatment.
Behavioural evaluation and tissue preparation
Mice were weighed twice a week. Behavioural trials for wob-
bler mice were done twice a week by the same operator blinded to
the treatment. Semi-quantitative score evaluation (paw and walking
abnormality) and quantitative measurements (running speed and
grip strength) were done, as per previous protocols (Mennini et al.,
2006; Mitsumoto et al., 1994). Grip strength was measured using a
Grip Meter apparatus (Ugo Basile, Comerio, VA, Italy).
Attheendofthetreatment,allanimalswere killed bytranscardiac
perfusion with 4% paraformaldehyde in 0.1 M PBS pH 7.4, under
deep anesthesia with Equithesin (1% phenobarbital/4% chloral
hydrate (vol./vol.),30 μl/10,i.p.).Immediatelyafter perfusion,biceps
muscles and spinal cord were rapidly removed and post-fixed for 4 h
in the same fixative (4 °C). All tissues were dehydrated and cryo-
protected with serial steps in 10%, 20% and 30% sucrose in 0.1 M
PBS at 4 °C until they sank and frozen in n-pentane at
biceps muscles and spinal cords were stored at 80 °C until analysis.
45 °C. The
For Nissl staining, cryostatic sections of cervical spinal cord
(C2 C6) were serially cut (30 μm thick) and processed by the
method reported by our group (Bigini et al., 2006). Five mice per
experimental group were used for motor neuron counting. At least
50 sections of cervical spinal cord were evaluated for each animal;
healthy motor neurons were counted in one side of each section.
The mean motor neuron number was calculated for each animal
and used for statistical analysis. Nissl-stained positive neurons
were counted by the same operator, blinded.
Quantification of gliosis
CD11b- (microglia) and GFAP- (astrocytes) positive cells were
measured by the procedure described by Park and colleagues (Park
et al., 2003), with slight modification. Briefly, for rhTBP-1-treated
(n=4) and vehicle-treated wobbler mice (n=4), 25-μm-thick
cervical sections were collected, then processed for CD11b and
GFAP immunostaining. At least ten sections were stained for
GFAP and for CD11b of each animal analyzed.
Sampling fields from the ventral column, nearly posterior to the
lateral part of lamina IX (Fig. 6A), were taken on a 40× field
objective; the sampling field was 265 μm×178.7 μm rectangle for
both GFAP and CD11b (Figs. 6B, C). Since neither GFAP nor
CD11b immunoreactivity was found in the grey matter of healthy
mice, our comparison was limited to the two affected groups.
Olympus-DB software, coupled to the microscope camera, was
used to count of astrocytes and microglial cells.
Immunohistochemistry and immunofluorescence
Immunohistochemical and immunofluorescence studies for
basic characterization included four 4-week-old and four 9-week-
old wobbler mice and the same number of age-matched control
mice. Cryostatic free-floating sections (30 μm thick) were cut in
the transverse plane through the cervical region of the spinal cord,
then processed. Immunostaining for GFAP (mouse monoclonal
antibody, Immunological Science; dilution 1:1000) and CD11b
(mouse monoclonal antibody OX 42, Serotec; dilution 1:50) were
performed as previously described (Mennini et al., 2004).
Immunostaining for TNF-α (mouse monoclonal antibody, HyCult
biotechnology; dilution 1:200), TNFR1 (mouse monoclonal anti-
body, HyCult biotechnology; dilution 1:200) and TNFR2 (mouse
monoclonal antibody, HyCult biotechnology; dilution 1:200) was
as previously described (Bigini and Mennini, 2004).
In order to avoid false-positive results, in control experiments,
the primary or secondary antibodies were used alone. These ex-
periments did not produce staining in any tissues.
For co-localization experiments, sections were processed as
described by Veglianese et al. (2006). The following markers were
analyzed: GFAP (mouse monoclonal antibody, Immunological Sci-
ence; dilution 1:1000), CD11b (rat monoclonal antibody OX 42,
Serotec; dilution 1:300), TNF-α (mouse monoclonal antibody, Hy-
466P. Bigini et al. / Neurobiology of Disease 29 (2008) 465 476
Cult biotechnology; dilution 1:200), TNFR1 (mouse monoclonal
antibody, HyCult biotechnology; dilution 1:50), TNFR2 (mouse
monoclonal antibody, HyCult biotechnology; dilution 1:50), P-JNK
(P-JNK(G-7) mouse monoclonal antibody, Santa Cruz; dilution
1:200), P-p38MAPK antibody (Phospho p38 (Thr 180-Tyr 182),
rabbit polyclonal antibody Cell Signaling Technology, dilution
Secondary antibodies (Alexa-488, Molecular Probes, 1:1000;
Alexa-Cy5-conjugated, 1:1000, Molecular Probes) were incubated
were incubated 30 min with 530 615 NeuroTrace Fluorescent Nissl
reagent (Molecular Probes, 1:100) at room temperature. To exclude
cross-reactions between the different antibodies, primary antibodies
were incubated with the secondary antibody associated with the
other primary antibody, but we found not signal for either antibody.
Sections were observed with an Olympus Fluoview microscope
BX61 with confocal system FV500. Images were pseudocolored
and the signals from the three different channels were automatically
merged by Olympus Fluoview software.
Western blot analysis
The levels of p38MAPK and JNK and their phosphorylated
forms were evaluated in the cervical and lumbar spinal cord of four
early symptomatic wobbler mice and four healthy littermates, by
Western blot analysis. Briefly, spinal cords were removed, rapidly
homogenized in ice-cold lysis buffer (Bonny et al., 2001) using a
manual Potter apparatus and then centrifuged at 13,000 rpm for
20 min at 4 °C. Supernatants were collected and protein concen-
trations determined. Samples were stored at
SDS polyacrylamide gel electrophoresis and transferred to a PVDF
using anti-P-JNK antibody (Cell Signaling Technology, dilution
1:2000), anti-JNK antibody (Cell Signaling Technology, dilution
1:2500), anti-P-p38MAPK antibody (Cell Signaling Technology,
dilution 1:1000), and anti-tubulin antibody (Santa Cruz Biotechnol-
ogy, dilution 1:50000). The blots were all normalized to tubulin and
The same experimental procedure was used to evaluate the
effect of rhTBP-1 on p38MAPK and JNK and their phosphorylated
forms. Twelve wobbler mice and twelve healthy littermates were
randomly enrolled to receive either rhTBP-1 or vehicle. Treatment
started at the 4th week of life and lasted 3 weeks, following the
schedule described earlier.
80 °C until use.
To see whether rhTBP-1 reached the central nervous system
(CNS), we measured rh-TNFR1 in plasma, cerebrospinal fluid
(CSF), brain and spinal cord of healthy mice. Twelve 4-week-old
was treated three times every other day with rhTBP-1, using the
schedule previously described. The second group received the same
volume of vehicle. Three hours after the last injection, mice were
anesthetized intraperitoneally with chloral hydrate (350 mg/kg), and
5 10 μl of CSF was drawn from the cisterna magna using a glass
capillary with a ~300-μm tip. Surgery was done very carefully to
avoid blood contamination. The mice were then killed, blood was
collected and the plasma isolated and frozen until use; brain and
spinal cord were immediately removed, frozen on dry ice and stored
at 80 °C. Tissue was homogenized in 10 volumes of ice-cold
phosphate-buffered saline (PBS) using an Ultra Turrax, clarified by
by a commercial ELISA kit, according to protocols described by the
supplier (Quantikine, human sTNFR1, R&D Systems, USA); the
lowest detectable concentration was 0.77 pg/ml. This assay
recognizes both natural and recombinant human sTNFR1.
Data calculation and statistical analysis
All data were expressed as mean±S.D.. Behavioural test results
were compared using two-way ANOVA with Bonferroni post-test.
Biceps weights and number of motor neurons were analyzed by
one-way ANOVA with Bonferroni post-test. The unpaired Stu-
dent's t-test was used for the rate of p38MAPK/P-p38MAPK and
JNK/P-JNK and the mean number of GFAP- and CD11b-positive
cells in vehicle- and rhTBP-1-treated wobbler mice. P values
b0.05 were considered significant. All statistical analyses were
done using the GraphPad Prism version 4.00 for Windows (Graph-
Pad Software, San Diego, CA, USA).
Characterization in early symptomatic wobbler mice
In 4-week-old wobbler mice, CD11b immunohistochemistry
found markedly more activated microglial cells than in healthy
littermates. CD11b immunoreactivity in the cervical spinal cord was
mainly in the whole grey matter and, to a lesser extent, in the white
matter (Fig. 1C,D). Unaffected littermates showed very weak
anterior horn from early symptomatic wobbler mice clearly in-
In this area, there were single CD11b-positive cells with a star-like
morphology, composed of thin, ramified processes, and some cells
assembled as clusters. These cells seemed highly concentrated in
specific areas, maybe close to degenerating motor neurons. This
morphological difference might be due to a further process of mic-
roglial differentiation toward a phagocytotic phenotype, in order to
permit the removal of neural debris from the injured area.
Like for CD11b immunostaining, sections of cervical spinal
cord from 4-week-old healthy mice faintly stained for TNF-α
(Figs. 1E, F) but cervical spinal cord of age-matched wobbler mice
were intensely immuoreactive (Fig. 1G,H). It is difficult to define
the localization of TNF-α by single immunostaining, but it seemed
mainly expressed in large neurons of the anterior horn (Fig. 1H).
No immunoreactivity for TNFR1 was detected in the cervical
wobbler mice showed an overall increase of immunoreactivity for
TNFR1 in the ventral horn (Fig. 1K). At higher magnification, ventral
exclusively, in large neurons of the anterior horn (Fig. 1L).
Immunohistochemistry for TNFR2 was done, but the staining
was almost undetectable in both 4-week-old controls and wobbler
mice (not shown).
Double staining experiments were carried out to verify the ac-
tual localization of TNF-α and its cognate receptors in the anterior
horn of the cervical spinal cord of four wobbler mice. We also
evaluated the activation of the two principal stress kinases (P-JNK
and P-p38MAPK) related to TNFR activation in the cervical and
467P. Bigini et al. / Neurobiology of Disease 29 (2008) 465 476
lumbar spinal cord of 4- and 9-week-old wobbler mice and their
healthy littermates. The bottom line of Fig. 2, panels A D, shows
the expression of TNF-α, TNFR1 and their associated MAPKs in
Nissl stained neurons in the anterior horn of cervical spinal cord of
early symptomatic wobbler mice. Double-staining experiments
confirmed the high expression of TNF-α (green) in Nissl-positive
neurons (red) in the wobbler mice (Fig. 2A). TNF-α and Nissl
staining merged mainly in large neurons from the cervical spinal
and in the controls. In addition, TNF-α was expressed not only in
neurons but also in other cells with different morphology (green).
TNFR1 immunoreactivity (green) was found in proximity to
Nissl-stained large neurons (red) exclusively in the anterior horn of
early symptomatic wobbler mice (Fig. 2B). TNFR1 expression was
not confined to Nissl-stained neurons but was found in other cells,
with a different morphology, too. In contrast to TNF-α expression,
TNFR1 immunoreactivity was detectable only in the periphery of
Nissl-stained neurons. Large neurons (red) in the anterior horn of the
cervical spinal cord from early symptomatic wobbler mice also
showed marked expression of P-JNK (green) (Fig. 2C). In these
out the cell body but not in the nucleus. In contrast to P-JNK, there
of TNF-α, TNFR1, P-JNK and P-p38MAPK was not confined to
neurons, in double-staining experiments we used specific markers
for activated microglia and astrocytes. The second line of Fig. 2,
panels E H, shows the expression in CD11b-positive cells of the
different markers of neuroinflammation in the cervical spinal cord
of early symptomatic wobbler mice. Co-localization experiments
showed that a large number of CD11b-positive cells (green) were
immunoreactive for TNF-α (red), the merging of the signals pro-
duced by the two antibodies (yellow) demonstrating the expression
of TNF-α in microglial cells (Fig. 2E). This representative picture
also shows that the staining for TNF-α was not confined to mic-
roglial cells, since there were also red-stained cells which did not
overlap with green-stained cells (arrowheads). TNFR1 immunor-
eactivity (red) co-localized with microglial cells (green) and other
cells not positive for CD11b (Fig. 2F). P-JNK immunoreactivity
(Fig. 2G). A clear expression of P-p38MAPK (red) in CD11b-
positive cells (green) was found in the anterior horn of early symp-
tomatic wobbler mice (Fig. 2H). However, whereas P-JNK was
found in single microglial cells still showing the typical star-like
morphology, the expression of P-p38MAPK seemed mainly con-
centrated in microglial nodules (merged area).
The third line of Fig. 2, panels I L, shows double-staining
experiments with a marker of activated astrocytes (GFAP) and with
the markers of neuroinflammation tested above. Like CD11b, GFAP-
positive cells (red) from early symptomatic wobbler mice expressed
TNF-α (green) (Fig. 2I). In contrast to CD11b-positive cells, which
express TNFR1, GFAP-positive cells (red) did not show any co-
localization with TNFR1 (green) (Fig. 2J).
To assess, the expression of stress kinases in astrocytes, co-
localization experiments were carried out using GFAP antibody and
P-JNK or P-p38MAPK antibodiesin cervical sectionsfrom 4-week-
there was no co-localization between astrocytes (red) and immunor-
Fig. 1. Representative pictures showing the pattern of staining for the main markers of neuroinflammation in control and early symptomatic wobbler mice, both
200 μm; (B, D, F, H, J, L)=40 μm.
468 P. Bigini et al. / Neurobiology of Disease 29 (2008) 465 476
of large anterior neurons. Double staining to reveal both GFAP-
positive cells (green) and P-p38-positive cells (green) showed co-
localization of this phosphorylated kinase in astrocytes of 4-week-
old wobbler mice (Fig. 2L).
Co-localization experiments in the cervical spinal cord of 9-week-
old wobbler mice showed a late expression of both TNFR1 and
phosphorylated JNK in activated astrocytes. While immunoreactivity
to the whole astroglial population (red) (Fig. 2P). There was a marked
increase of CD11b immunoreactivity (green) close to the degenerating
neurons (red) in the anterior horn of the cervical spinal cord of late
symptomatic wobbler mice (Fig. 2N). Although activation of
microglial cells was significantly stronger in 9-week-old wobbler mice
in cervical sections of 4-week-old healthy mice and in the lumbar
region of symptomatic mice. However, except for Nissl staining, they
did not produce appreciable immunostaining (not shown).
To confirm that the high immunoreactivity for P-JNK and
P-p38MAPK were actually due to an increase in the rate of phos-
phorylation rather than to constitutive overexpression of JNK and
p38MAPK, Western blot analysis was done on cervical spinal cord
homogenates from 4-week-old wobbler mice and healthy litter-
mates. There were no real differences in JNK and p38MAPK ex-
Fig. 2. (A–L)Representativepictures showing patternofexpression theofTNF-α (first column),TNFR1 (second column), P-JNK (thirdcolumn) and P-p38MAPK
4-week-old mice. (M–O) Representative pictures showing TNFR1 immunostaining in the cervical spinal cord region of 9-week-old mice in the presence of GFAP-
spinal cord region of 9-week-old mice. Scale bar: (A–C)=40 μm; (D)=60 μm; (E–H)=80 μm; (I–P)=60 μm.
469P. Bigini et al. / Neurobiology of Disease 29 (2008) 465 476
pression (Fig. 7A). The immunoblot of P-PJNK and P-p38MAPK
confirmed the immunofluorescence findings (Fig. 7A), showing
almost selective expression of the two phosphorylated proteins
in the cervical spinal cord of symptomatic wobbler mice. Lumbar
tracts from wobbler and control mice did not differ in the ex-
pression of JNK and p38MAPK and there were no increases in the
phosphorylated forms in affected mice (not shown).
The mean body weight of wobbler mice was lower than the
healthy littermates, this being characteristic of the animal model, as
previously reported. No differences were detected in the growth
rate of treated and untreated mice, indicating that the drug was well
tolerated for the period of treatment (not shown).
Vehicle-treated wobbler mice showed rapid, progressive wors-
ening of paw and walking abnormalities, resulting in higher scores.
rhTBP-1 had a beneficial effect improving the walking and paw
abnormality scores, which were lower than those of vehicle-treated
wobbler mice (Figs. 3A, B); the effect was significant (pb0.001)
on both the semi-quantitative scores. Behavioral data for control
mice treated with rhTBP-1 are not shown since there were no
differences in paw position or walking compared to vehicle-treated
controls (score=0, normal).
The running speed of healthy mice was not significantly modi-
fied by treatment, values ranging from 20 to 40 cm/s. However, the
running speed progressively declined throughout the whole treat-
ment time in vehicle-treated and rhTBP-1-treated wobbler mice.
However, the progressive worsening was significantly reduced by
rhTBP-1 starting from the 2nd to 3rd week of treatment (Fig. 3C).
Results were similar for the normalized grip strength. Control mice
showed greater grip strength in relation to their increase both in
muscle strength and in body weight. Therefore, the values were
normalized considering each animal s body weight. Healthy mice
had normalized grip strength ranging from 3.5 to 5 g/g body weight
at 4 and 9 weeks. Like running speed, rhTBP-1- and vehicle-treated
Fig. 3. Behavioural tests in wobbler mice treated with rhTBP-1. rhTBP-1 was given s.c. at the dose of 10 mg/kg, three times a week; treatment was carried out
from the 4th to the 9th week of age. (A) walking abnormality, (B) paw abnormality; (C) running speed (cm/s); (D) grip strength (normalized to body weight).
Each point represents the mean±S.D. of 8 mice. Statistical analysis: pb0.001 for the overall treatment (two-way ANOVA);⁎pb0.05,⁎⁎pb0.01,⁎⁎⁎pb0.001
different from vehicle (Bonferroni post-test).
470 P. Bigini et al. / Neurobiology of Disease 29 (2008) 465 476
wobbler mice showed a progressive loss of the normalized grip
strength but already after the first week of treatment there was
significant improvement compared to untreated mice (Fig. 3D).
Behavioral data for control mice treated with rhTBP-1 are not
shown because grip strength and running speed were the same
detected during treatment as in vehicle-treated healthy mice.
The rate of motor neuron loss in 9-week-old vehicle-treated and
rhTBP-1-treated wobbler mice is reported in relation to the numbers
of motor neurons in healthy littermates (Fig. 4A). Vehicle-treated
wobbler mice had significantly fewer motor neurons than compared
to healthy littermates, being controls (pb0.001). The number of
motor neurons in rhTBP-1-treated wobbler mice was signifi-
cantly higher than in vehicle-treated mice (vehicle 4.7±0.1;
rhTBP-1 6.9±0.5; pb0.05). There were no differences between
vehicle-treated and rhTBP-1-treated controls (vehicle 15.7±0.9;
rhTBP-1 15.9+1.1). Nissl staining indicated that motor neurons
from rhTBP-1-treated wobbler mice had an intense, homogeneous
in vehicle-treated wobbler mice the small number of Nissl-positive
motor neurons was only faintly stained and showed substantial
changes in cell body shape, very likely due to sustained chro-
matolysis (Fig. 4D).
Fig. 4. Neuropathological effects of rhTBP-1 treatment. (A) Percentage of motor neuron in cervical spinal cord, (B) percentage of biceps weight in wobbler mice
treated with rhTBP-1 or vehicle. Data are reported as percentage of the average values in control mice and expressed as mean±S.D.; n=5 mice for each
experimental group (see Materials and methods).⁎⁎⁎pb0.001 different from wobbler vehicle. One-way ANOVAwith Bonferroni post-test. Lower panel: Nissl
staining in the lamina IX (central column motor neurons) in vehicle-treated healthy mice (C), vehicle-treated wobbler mice (D) and rhTBP-1-treated wobbler
mice (E). Scale bar: 40 μm.
Fig. 5. Representative pictures showing the pattern of staining for the main markers of neuroinflammation in the anterior horn of cervical spinal cord in 9-week-
old vehicle-treated wobbler mice (A–D) and age-matched rhTBP-1-treated wobbler mice (E–H). Scale bar: 50 μm.
471P. Bigini et al. / Neurobiology of Disease 29 (2008) 465 476
Consistent with the smaller loss of cervical motor neurons after
rhTBP-1, biceps weight, an indirect measure of the degree of at-
rophy, was significantly higher in rhTBP-1-treated wobbler mice
than vehicle-treated wobbler mice (vehicle 4.2±0.5 mg; rhTBP-1
5.9±0.4 mg; pb0.05). Like motor neuron loss, biceps weight in
mice (vehicle 14.5±1.1 mg; rhTBP-1 15.6±1.6 mg).
Comparing the pattern of GFAP immunostaining in 9-week-old
vehicle-treated and rhTBP-1-treated wobbler mice, small reduction
in the main markers of glial activation were detectable in terms of
astrocyte hypertrophy and distribution and localization of activated
astrocytes acquired hypertrophic features with larger cell bodies and
thicker processes, GFAP-positive cells were significantly fewer
rhTBP-1-treated (36.7±2.7, n=4) than in vehicle-treated wobbler
mice (45.4±4.1, n=4) (pb0.02, Figs. 6B D).
Positive CD11b staining was observed in rhTBP-1 and vehicle-
treated wobbler mice at 9 weeks (Figs. 5B, F). In correspondence to
lamina IX, close to motor neurons, activated microglial cells were
markedly enlarged: cell bodies swelled and acquired a round shape,
losing the typical distal branches. Although rhTBP-1 did not fully
arrest microglial activation, CD11b-positive cells in rhTBP-1-treated
wobbler mice (18.6±2.6, n=4) were significantly lower than in
In 9-week-old wobbler mice, after 5 weeks of treatment with
rhTBP-1 or vehicle, immunoreactivity for TNF-α and TNFR1 was
TNF-α immunostaining, rhTBP-1-treated wobbler mice (Fig. 5G)
even had more intense immunoreactivity than vehicle-treated mice
(Fig. 5C). However, this difference might be due to the larger
number of surviving motor neurons in the rhTBP-1-treated group.
The pattern of TNFR1 staining in the anterior horn of vehicle-
treated mice confirmed the overexpression of this receptor in neu-
ronal cell bodies and in thinner cells showing the typical glial
morphology, mainly close to affected motor neurons (Fig. 5D).
rhTBP-1-treated mice showed a similar pattern of TNFR1 immu-
noreactivity (Fig. 5H) with fewer cells immunoreactive for TNFR1
with glial morphology, close to motor neurons. The difference may
be due to the lower reactive gliosis in the ventral horn of wobbler
mice treated with rhTBP-1 because of the less marked reduced
CD11b, TNF-α and TNFR1 staining were almost absent in
both groups of control mice and the sections were similar to those
in 4-week-old healthy mice (Figs. 1A, B, E, F, I, J). rhTBP-1 did
not modify the pattern of immunoreactivity for GFAP and TNFR2,
which was almost absent in both groups.
The ratio of the phosphorylated and the non-phosphorylated
forms of JNK and p38MAPK was measured in the cervical region
of wobbler mice after 3 weeks of TBP-1 treatment, compared to
vehicle-treated wobbler mice. The constitutive expression of JNK,
p38MAPK and their phosphorylated forms was the same in 7- and
4-week-old healthy mice.
Fig. 7 shows Western blot findings for P-JNK, JNK, P-
p38MAPK, p38MAPK and tubulin in the cervical region. Although
P-JNK/JNK ratio in rhTBP-1 wobbler mice (1.03±0.28) was not
significantly lower than in vehicle-treated wobbler mice (1.46
±0.35), rhTBP-1-treated mice showed a marked tendency to lower
P-JNK (Figs. 7B, D).
rhTBP-1 had a stronger effect in wobbler mice in inhibiting
p38MAPK phosphorylation. p38MAPK and P-p38MAPK levels in
treated and untreated groups (Figs. 7C, E) indicated that rhTBP-1
significantly lowered the P-p38MAPK/p38MAPK ratio (0.18±
0.02) compared to vehicle-treated mice (0.36±0.08, pb0.03).
Fig. 6. The number of GFAP- and CD11b-positive cells have been quantified by sampling a rectangular area behind the lateral part of lamina IX (A).
Representative microphotographs of GFAP- and CD11b-positive cells inside the rectangle are shown in panels B and C, respectively. (D, E) Bar graph showing
the mean number of GFAP- and CD11b-positive cells in rhTBP-1- and vehicle-treated wobbler mice, respectively. Data are expressed as mean±S.D.; n=4 mice
for each experimental group (see Materials and methods).⁎pb0.02 different from vehicle-treated wobbler mice (unpaired Student's t-test). Scale bar:
(A)=250 μm; (B, C)=20 μm.
472P. Bigini et al. / Neurobiology of Disease 29 (2008) 465 476
expression of JNK and p38MAPK or their rate of phosphorylation,
which remained as low as that for vehicle-treated age-matched con-
trol mice (data not shown).
rhTBP-1 was detectable in brain and spinal cord homogenates of
healthy mice, although less than in plasma. The levels of rhTBP-1 in
CSF were similar to those in brains and spinal cord of treated mice
(Table 1). In animals receiving vehicle alone, rhTBP-1 was below the
minimum detectable concentration (0.77 pg/ml) and, in both experi-
mental groups, no significant cross-reactivity or interference was
observed for rhTNF-α, rmTNF-α, rhTNF-β, rhsTNFR2, rmTNFR1,
TNF- - and TNFR1-associated pathway in early symptomatic
The first evidence from this study is that in the anterior horn of
the cervical spinal cord of early symptomatic wobbler mice, motor
neuron death and glial activation is accompanied by overexpression
of TNF-α and its cognate receptor TNFR1. In contrast to other mo-
et al., 2006) and axonal degeneration (George et al., 2005), we did
not find a parallel increase of TNFR2 in early symptomatic
wobbler mice. In sporadic ALS patients, the gene expression profile
studies in total homogenate of lumbar spinal cord (Malaspina et al.,
2001) or in laser-dissected spinal motor neurons (Jiang et al., 2005)
showed no changes of TNF-α. This difference from mouse models
might be explained bythe fact thatexperimental samples were taken
at autopsy from ALS patients, when a large number of motor
neurons are already lost. However, in the study on laser-dissected
associated with TNF-α were overexpressed (Jiang et al., 2005).
We found that TNF-α/TNFR1 overexpression was accompanied
by selective hyperphosphorylation of JNK and p38MAPK, with no
concomitant increase of JNK and p38MAPK expression. JNK and
p38MAPK activation is considered a common step of neuronal
injury and can be activated by several upstream stimuli (Borsello
Fig. 7. (A) Representative immunoblots from cervical spinal cord homogenatesfrom 4-week-old control and wobbler mice processed by using antibodiesagainst
JNK, p38MAPK, P-JNK, and P-p38MAPK. (B, C) Cervical spinal cord homogenates from wobbler mice after 3 weeks of treatment with rhTBP-1 (left) and
vehicle (right), blotted by using antibodies against P-JNK (B, upper lane), JNK (B, middle lane), P-p38MAPK (C, upper lane), p38MAPK (C, middle lane), and
tubulin (B, C, lower lanes). (D, E) Histograms representing the ratio between P-JNK and JNK (D) and the ratio between P-p38MAPK and p38MAPK (E) in
rhTBP-1- and vehicle-treated wobbler mice. Data are expressed as mean±S.D.; n=6 mice for each experimental group (see Materials and methods).⁎pb0.02
different from vehicle-treated wobbler mice (unpaired Student's t-test).
Evaluation of rhTBP-1 concentration (ng/ml or ng/g wet weight)
Tissue rhTBP-1 concentration (ng/ml or ng/g wet weight)
in healthy mice 3 h after the last administration of rhTBP-1 (10 mg/kg s.c.,
three dosesevery other dayfor a week).The signal detectedin CSF andCNS
regions of untreated mice was not different from blank values. Data are
expressed as mean±S.D. of 6 mice.
473 P. Bigini et al. / Neurobiology of Disease 29 (2008) 465 476
et al., 2003; Cahill et al., 1996; Sluss et al., 1994; Wessig et al.,
2005) and the downstream phosphorylation of these two proteins
after TNFR activation has been reported in many in vivo and in
vitro models of neurodegeneration (Raoul et al., 2002). Activation
of the JNK and p38MAPK pathway occurs in neurodegenerative
disorders such as Alzheimer's disease, Parkinson's disease, multiple
sclerosis, and ischemia, and inhibition of JNK and p38MAPK ac-
tivity confers protection against neuronal death, reducing brain
injury (Barone et al., 2001; Harper and Wilkie, 2003).
Cellular localization of TNF- - and TNFR1-associated pathway
activation in early symptomatic wobbler mice
While early symptomatic wobbler mice TNF-α showed over-
expression in all the cellular populations of the cervical spinal cord, in
our double-staining experiments, TNFR1, P-p38MAPK and P-JNK
cells, in accordance with the results obtained in SOD1G93A and
SOD1G37R mice (Veglianese et al., 2006). GFAP-positive cells be-
in symptomatic mice. It is not clear why TNFR1, P-JNK and P-p38
were found in microglial cells and not in astrocytes of 4-week-old
wobbler mice. However, the two different types of glial cells may
respond differently to the environmental changes due to motor neuron
of motor neuron loss without delaying the onset of symptoms (Beers
et al., 2006; Boillee et al., 2006). In addition, a selective and transient
activation of different kinases linking TNFRs to JNK and p38MAPK
phosphorylation was clearly seen in the early phases of the disease in
SOD1 transgenic mice (Veglianese et al., 2006).
These results suggest that the neuroinflammatory pathway related
to microglial activation and TNF-α overexpression is not responsible
source of damage, thus accelerating the neurodegenerative effect.
The present findings, confirming the involvement of glial activation
and TNF-α expression already in the early phases of the disease
(Blondet et al., 1995; Boillee et al., 2001; Schlomann et al., 2000),
suggest that the activated microglia responds to TNF-α by overex-
pressing its cognatereceptor andby phosphorylatingboth P-JNKand
P-p38MAPK from the first phases of wobbler motor neuron disease.
The absence of glial activation, TNF-α/TNFR1 overexpression and
p38MAPK/JNKphosphorylation inthe lumbar regionof early symp-
tomatic wobbler mice, where motor neurons are conserved, suggests
an important role of glial-induced neuroinflammation in the cervical
However, although these results seem to confirm the detri-
mental effect of activated microglia in the cervical spinal cord of
early symptomatic wobbler mice, we cannot state that microglial
activation is the crucial event responsible for motor neuron death.
The link between microglial activation and motor neuron death is
still far form clear and a report that such activation and the
progressive increase in the expression of TNF-α and TNFR1 re-
ported in the spinal cord of mnd mice is not accompanied by an
apparent loss of motor neurons (Mennini et al., 2004) further
complicates our understanding of the problem.
Effect of rhTBP-1 treatment
To better investigate the involvement of TNF-α in motor neuron
death, early symptomatic wobbler mice were given a pharmacolo-
gical treatment aimed at buffering TNF-α levels, thus reducing
TNFR1 activation. rhTBP-1 significantly reduced neuromuscular
decayfrom thesecond weekand its effectiveness remained through-
out the investigation until 9 weeks of age, corresponding to the peak
tests, rhTBP-1-treated wobbler mice had significantly more cervi-
cal motor neurons and heavier biceps muscle than vehicle-treated
wobbler mice, similar to that after treatment with riluzole (Fuma-
galli et al., 2006). Although rhTBP-1-treated wobbler mice showed
a small but significant decrease in the number of positive astro-
cytes and microglial cells, there was no apparent reduction of either
TNF-α or TNFR1 immunoreactivity compared to vehicle-treated
slightly but significantly lower in rhTBP-1 than vehicle-treated
wobbler mice after 3 weeks of treatment. This suggests that the
protective effect is related to competition between the exogenous
rhTBP-1-soluble protein and the endogenous TNFR1 (clearly
overexpressed in cervical motor neurons already in the early symp-
tomatic phase of the wobbler motor neuron disease) rather than to a
reduction of TNF-α expression.
We found measurable levels of the drug in the nervous tissues of
mice but the concentration was considerably lower than in plasma,
plasma in the blood vessels rather than to the actual presence of
rhTBP-1 on tissues. The finding of a low concentration of rhTBP-1
alsointheCSF indicatesthatthisamountmight berelated eithertoa
passive diffusion from the plasmatic compartment ( 1%) or to
active, although low, transport across the blood CSF barrier.
The evidence that in both symptomatic wobbler mice and in
healthy littermates TNF-α plasma levels, measured by ELISA, were
below the threshold of sensitivity (data not shown), rendered ex-
tremely unlikely that rhTBP-1 acts peripherally by neutralizing cir-
culating TNF-α and therefore indirectly reducing the flux of TNF-α
from periphery to brain. However, in transgenic mice overexpres-
sing sTNFR1, motor neuron survival after axotomy of the facial
nerve correlated with sTNFR1 blood levels (Terrado et al., 2000),
that were 100 800 ng/ml, even lower than those found in our study.
The decreased rate of phosphorylation of the two main stress
kinases related to TNFR1, JNK and p38MAPK suggests a central
effect of the treatment on TNF-α related pathways.
Our study indicates that TNF-α and TNFR1 overexpression and
JNK/p38MAPK phosphorylation occur in early symptomatic mice,
so this activation may contribute to motor neuron damage. The
involvement of TNF-α was confirmed by the fact that rhTBP-1
reduced symptom progression, motor neuron loss, gliosis and JNK/
p38MAPK phosphorylation. At variance with this result, the si-
lencing of TNF-α gene in mutant mice expressing SOD1G37R or
SOD1G93A neither prolonged the life span nor reduced the rate of
motor neuron loss (Gowing et al., 2006). However, no reduction of
reactive gliosis was reported in that study. The toxicity of TNF-α
34 cell line co-cultured with an immortalized mouse microglial cell
line (BV-2) stimulated with LPS (He et al., 2002; Woo et al., 2003).
Therefore, the significant decrease in glial activation in the ventral
horn of wobbler mice treated with rhTBP-1 might be more than
be a factor contributing to the lowered effect of TNF-α itself.
474P. Bigini et al. / Neurobiology of Disease 29 (2008) 465 476
Interestingly, thalidomide, a potent antiinflammatory and im-
munomodulatory drug, whose effects include inhibition of TNF-α
synthesis, delays death in SOD1G93A mice (Kiaei et al., 2006).
In conclusion, our data strongly suggest an involvement of
the TNF-α/TNFR1 pathway in the amplification of motor neuron
death in wobbler mice and indicate that rhTBP-1 offers a signifi-
cant neuroprotective effect in this murine model of motor neuron
This work was partly supported by FIRB grant no.
sity and Scientific Research. The authors are grateful to Dr. Judy
Baggott for English style revision, Dr. Pia Villa and Dr. Sara Triulzi
for the measurements of plasma TNF-α and also we wish to thank
Mr. Felice De Ceglie and Mr. Alessandro Soave for the technical
support in preparing the figures.
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