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Parvalbumin and calbindin D-28k immunoreactivity in transgenic mice with a G93A mutant SOD1 gene
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Parvalbumin and calbindin D-28k immunoreactivity in transgenic mice with a G93A mutant SOD1 gene Shoichi Sasaki a,⁎, Hitoshi Warita b , Takashi Komori c , Tetsuro Murakami d , Koji Abe d , Makoto Iwata a a
Department of Neurology, Neurological Institute, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan Department of Neurology, Yonezawa National Hospital, Japan c Department of Clinical Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Japan d Department of Neurology, Okayama University Medical School, Japan b
A R T I C LE I N FO
AB S T R A C T
Article history:
Immunohistochemical study was performed to examine if calcium-binding proteins are
Accepted 28 January 2006
involved in the degeneration of motor neurons in the brain stems and the spinal cords of
Available online 20 March 2006
transgenic mice carrying a G93A mutant human SOD1 gene. Specimens from age-matched non-transgenic wild-type mice served as controls. In the spinal cord of the controls, the
Keywords:
density of parvalbumin-immunoreactive neurons was highest in the large anterior horn
Amyotrophic lateral sclerosis
neurons and lower in the posterior horn neurons in the spinal cord. On the other hand,
Transgenic mice
calbindin D-28k immunoreactivity was much less apparent than that observed with
SOD1 mutation
parvalbumin antisera. Rexed's lamina II was densely immunostained for calbindin D-28k,
Parvalbumin
whereas, in the anterior horn, calbindin-D-28k-positive small neurons were barely
Calbindin D-28k
dispersed in a scattered pattern. In transgenic mice, parvalbumin-positive anterior horn
Immunohistochemistry
neurons were severely reduced, even at the presymptomatic stage, whereas calbindinpositive neurons were largely preserved. At the symptomatic stage, both parvalbumin and calbindin D-28k immunoreactivity markedly diminished or disappeared in the anterior horn. Immunoblotting analysis revealed a significant reduction of immunoreactivity to parvalbumin antibody in transgenic mice compared with the controls. In the brain stem, parvalbumin-positive oculomotor and abducens neurons and the calbindin D-28k-positive sixth nucleus were well-preserved in transgenic mice as well as in the controls. Thus, the diffuse and severe loss of parvalbumin immunoreactivity of large motor neurons even at early stages in SOD1-transgenic mice and the absence of calbindin D-28k immunoreactivity of normal large motor neurons suggest that these calcium-binding proteins may contribute to selective vulnerability and an early loss of function of large motor neurons in this SOD1transgenic mouse model. © 2006 Elsevier B.V. All rights reserved.
⁎ Corresponding author. Fax: +81 3 5269 7324. E-mail address: [email protected] (S. Sasaki). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.01.129
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1.
197
Introduction
Parvalbumin and calbindin D-28k are calcium-binding proteins that appear to be involved in the buffering of free intracellular calcium, and they play an important role in regulating neuronal calcium homeostasis (Baimbridge et al., 1992; Heizman and Braun, 1992). It has been suggested that the selective neuronal distribution of calcium-binding proteins may be a determinant of vulnerability to excitotoxicity, in which a disturbance of intracellular calcium homeostasis plays a major role (Shaw, 1994). A number of studies suggested that at least two factors are considered to contribute to selective vulnerability of motor neurons: the high density of Ca-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type of glutamate receptors (Carriedo et al., 1996; Ikonomidou et al., 1996; Rothstein et al., 1993) and the low expression of the calcium-binding proteins parvalbumin and/or calbindin D-28k in motor neurons (Celio, 1990; Ince et al., 1993). However, little is known about immunoreactivity of calcium-binding proteins in mutant SOD1-transgenic mice (Knirsch et al., 2001; Morrison et al., 1996; Nimchinsky et al., 2000; Siklos et al., 1999), especially parvalbumin immunoreactivity in G93A mutant SOD1-transgenic mice. In this study, we performed immunohistochemical investigation in order to determine whether or not these calcium-binding proteins are involved in the degeneration of motor neurons in transgenic mice carrying a G93A mutant human SOD1 gene from presymptomatic to symptomatic stages.
Fig. 1 – Many anterior horn neurons are immunostained for parvalbumin (control case, 24 weeks, lumbar spinal cord). The somata of anterior horn neurons and neuronal processes are immunoreactive.
2.
Results
2.2.
2.1.
Non-transgenic littermates
At the age of 24 weeks (early presymptomatic), all levels of the spinal cord were free from neuronal depletion in the anterior horns. Parvalbumin immunoreactivity in the anterior horns was diminished at the lumbar level (Fig. 4) but was well-preserved at the cervical level. There was no difference in calbindin D-28k immunoreactivity between controls and transgenic mice. At the age of 28 weeks (late presymptomatic), slight neuronal loss of anterior horn cells (those exceeding 35 μm in the mean of long and short diameters) was recognized at the cervical (28.0 ± 3.4) and lumbar (27.6 ± 5.2) levels, showing significantly lower values than those of the controls (36.1 ± 2.9, 35.4 ± 2.8, respectively) (P < 0.01). There were prominent vacuolar changes of various sizes in the neuropil of the anterior horns and anterior roots in the anterior column. Lewy-body-like inclusions were occasionally observed in the neuropil of the anterior horns. Parvalbumin-positive anterior horn neurons were much more reduced at all levels of the spinal cord, as compared with the early presymptomatic stage, and parvalbumin-positive posterior horn neurons also decreased in number, though to a lesser extent. There was no significant reduction in the number of calbindin D-28k immunoreactive neurons. At the age of 32 weeks (early symptomatic stage), the anterior horns showed a moderate neuronal loss of anterior horn cells at the cervical (21.8 ± 11.7) and lumbar (25.4 ± 4.6) levels; these values were significantly lower than those obtained with the controls (37.4 ± 2.2, 32.9 ± 5.2, respectively) (P < 0.01). Vacuolar
Parvalbumin immunostaining in the spinal cord was much more intense than that observed with calbindin D-28k antisera. Both the gray and white matter of all segments of the spinal cord showed parvalbumin immunoreactivity. In the gray matter, the perikarya and neuronal processes of many small to large anterior horn neurons, and of neurons of intermediate gray (lamina VII), and, to a lesser extent, of neurons in the posterior horn (Rexed's laminae II–VI), were positively immunostained for parvalbumin (Fig. 1). The density of parvalbumin-immunoreactive neurons was highest in the large anterior horn cells. Some neurons around the central canal (lamina X) and in Clarke's nucleus also exhibited positive immunoreactivity for parvalbumin. In the white matter, axons of all funiculi were diffusely immunostained. Parvalbumin-positive axons were regularly seen in the ventral root, as well as in the dorsal root. On the other hand, many small cells, together with their processes, in the posterior horn (Rexed's lamina II) were consistently immunoreactive with anti-calbindin D-28k antibody (Fig. 2). Calbindin-positive medium-sized or small neurons were less numerous and barely dispersed in a scattered pattern through the ventral horn; however, the large anterior horn cells were not immunoreactive (Fig. 3). Due to the low number of these neurons, it was difficult to assess the quantitative changes in calbindin D-28k immunoreactive neurons. All funiculi of the
white matter were not, or only weakly, immunostained for calbindin D-28k. The ventral root and the dorsal root were not immunostained. The blood vessels were not immunostained for parvalbumin or calbindin D-28k. Somata of the sixth and seventh cranial nerve nuclei were positively immunostained for calbindin D-28k, whereas perikarya and fibers of the third nucleus exhibited no immunoreactivity. On the other hand, oculomotor (the nucleus of Darkschewitsch), abducens and facial neurons were parvalbumin-positive.
Transgenic mice
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analysis of the band using Scion image revealed a significant reduction (31%) of immunoreactivity to parvalbumin antibody in transgenic mice compared with the controls (Student's t test, P < 0.01) (Fig. 8). On the other hand, there was no detectable expression of calbindin D-28k protein in the spinal cord of either the transgenic or the non-transgenic group.
3.
Fig. 2 – Many small cells in the posterior horn (Rexed's lamina II) are immunostained for calbindin D-28k (control case, 28 weeks, cervical spinal cord).
changes were observed in the neuropil of the anterior horns and the anterior roots, and Lewy-body-like inclusions were frequently demonstrated within the neuronal processes including the proximal axons. Parvalbumin immunoreactivity was markedly reduced in the anterior horns. Calbindin D-28k immunoreactivity was not observed in the anterior horns, whereas no differences were observed as regards the distribution and density of calbindin D-28k in the posterior horns of transgenic mice and the controls. At the age of 35 weeks (end-stage), severe neuronal loss of the anterior horn neurons accompanied by prominent astrogliosis was observed at the cervical (13.0 ± 4.6) and lumbar (7.0 ± 0.9) levels, showing significantly lower values than those observed in the controls (31.5 ± 6.8, 32.8 ± 2.5, respectively) (P < 0.001). Lewybody-like inclusions were frequently observed in the neuronal processes. However, vacuolar changes were less prominent in the anterior horns. Parvalbumin immunoreactivity was not shown in the anterior horns (Fig. 5) nor was calbindin D-28k-positive immunoreactivity seen in the anterior horns, while in the posterior horn (Rexed's lamina II), calbindin D28k immunoreactivity was well-preserved, as was observed in the controls. The immunoreactivity of parvalbumin and calbindin D-28k in the white matter was well-preserved, but it did decrease in the ventral root as well as in the dorsal root. The blood vessels were not immunostained for either protein. Neither the sections processed with omission of the primary antibodies nor those incubated with the preabsorbed antibodies at the late presymptomatic stage (28 weeks) showed any immunoreaction product deposits (Fig. 6). In the brain stem, calbindin D-28k immunoreactivity of the seventh nucleus was decreased as compared with the controls, whereas it was well-preserved in the sixth nucleus. Parvalbumin immunoreactivity was well-preserved in oculomotor (Fig. 7) and abducens neurons, although it was decreased in facial neurons.
2.3.
Discussion
In non-transgenic littermates of the controls, both parvalbumin and calbindin D-28k were specific for neurons and their processes and were not observed in the glial and supporting cells. The density of parvalbumin-immunoreactive neurons was highest in the large anterior horn neurons and lower in the posterior horn cells, whereas calbindin D-28k was predominantly expressed in the small anterior neurons (presumably interneurons) and the posterior horn (Rexed's lamina II). The distribution of parvalbumin and calbindin D28k immunoreactivity in the mouse spinal cords in this study was almost the same as that observed in rat spinal cords (Celio, 1990; Laslo et al., 2000; Lim et al., 2000; Ren and Ruda, 1994), although Zhang et al. reported parvalbumin and calbindin D-28k immunoreactivity in a few medium-sized (about 30–35 μm in diameter) and small (about 15–18 μm in diameter) neurons in the ventral horn, calbindin D-28kpositive neurons in the superficial layers of the posterior horn and parvalbumin-positive neurons in layers II–III of the dorsal horn of the rat spinal cord (Zhang et al., 1990). On the other hand, in the human spinal cord, parvalbumin is confined to a few interneuronal processes in the ventral horn only, while calbindin is present in many interneurons
Immunoblotting
Quantitative Western blot analysis of extracted mice lumbar spinal cord protein for parvalbumin showed a band corresponding to parvalbumin protein (12 kDa) in both the transgenic and non-transgenic groups. Then, quantitative
Fig. 3 – Calbindin-positive small-sized neurons are dispersed in a scattered pattern in the anterior horn, but large anterior horn cells are not immunostained (control case, 24 weeks, lumbar spinal cord).
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199
Fig. 4 – (A) At the age of 24 weeks (early presymptomatic stage), parvalbumin immunoreactivity is diminished in the anterior horn in the lumbar spinal cord. (B) Age-matched control case for comparison.
and fibers within the gray matter (Ince et al., 1993). However, lower motor neurons at all spinal levels are negatively immunostained for both antibodies (Ince et al., 1993). Thus, the distribution of immunoreactivity of these calcium-binding proteins in the mice or rat spinal cords is different from that of the human (Celio, 1990), as was confirmed by this study. The functions of parvalbumin and calbindin are not still clear, but a number of reports suggest that these proteins may protect cells by binding and buffering increased intracellular
calcium (Chard et al., 1993; Heizman and Braun, 1992; Lledo et al., 1992; Mattson et al., 1991; Miller, 1991). Selective vulnerability of motoneurons to an increased calcium load may be related to their low expression of the calcium-binding proteins calbindin D-28k and/or parvalbumin (Celio, 1990). Excitotoxic processes are involved in motoneuron degeneration, whereby overstimulation by glutamate results in elevated levels of intracellular calcium (Shaw and Eggett, 2000), which in turn initiates a cascade of destructive metabolic processes such as
Fig. 5 – (A) At the age of 35 weeks (end-stage), almost no parvalbumin immunoreactivity in the anterior horn can be seen (lumbar spinal cord). (B) Age-matched control case for comparison.
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Fig. 8 – Western blot analysis shows a band corresponding to parvalbumin (12 kDa) in both transgenic and non-transgenic groups, and immunoreactivity to parvalbumin antibody in transgenic mice was significantly reduced by 31% compared with the controls (Student's t test, P < 0.01).
Fig. 6 – The section incubated with the preabsorbed antibody shows that the parvalbumin immunostaining was completely prevented (lumbar spinal cord). the activation of Ca2+-dependent protein kinases, endonucleases and phospholipases (Choi, 1988; Nicotera and Orrenius, 1992). Increased intracellular calcium has been shown to accelerate the production of nitric oxide and superoxide (Coyle and Puttfarcken, 1993; Dawson et al., 1994; Lafon-Cazal et al., 1993), free radicals that are precursors to both peroxynitrite (Beckman and Crow, 1993) and hydrogen peroxide (Fridovich, 1974). In amyotrophic lateral sclerosis (ALS), calcium is increased within neurons and motor nerve terminals of biopsied human muscle specimens, as well as in motor nerve terminals of mice following passive transfer of ALS immunoglobulin G (IgG) (Krieger et al., 1994; Pullen and Humphreys, 2000; Siklos et al., 1996). Calcium is also increased in vesicular structures within spinal motoneurons of trans-
Fig. 7 – At the end-stage, parvalbumin immunoreactivity in the accessory oculomotor nucleus (the nucleus of Darkschewitsch) is well-preserved. Scale bars, Figs. 1–3, 7): 50 μm, Figs. 4, 5: 125 μm, Fig. 6: 200 μm.
genic mice expressing mutant human SOD1 (Siklos et al., 1998). In human autopsy specimens, motoneurons relatively deficient in the calcium-binding proteins (e.g. spinal and hypoglossal motoneurons) are lost early in ALS, whereas motoneurons expressing high levels of these proteins (cranial nerves III, IV, VI and Onuf's nucleus motoneurons) are relatively spared (Alexianu et al., 1994; Elliot and Snider, 1995; Ince et al., 1993) that is true in mutant SOD1-transgenic mice in this study. These results suggest that cells containing these calcium-buffering proteins may be less vulnerable to the pathological process associated with motor neuron disease and that calcium-binding proteins play a role in protecting neurons from degeneration in ALS. However, the role of calcium-binding proteins in establishing this differential vulnerability of motoneurons to degeneration has been questioned by recent observations that vulnerable spinal motoneurons express parvalbumin (Philippe et al., 1993), which is consistent with the results of the present study. A recent demonstration of reduced SOD1 activity may implicate motoneuron damage through changes in motoneuron calcium homeostasis since intracellular calcium chelators were shown to prevent free-radical-induced DNA breakdown and cytotoxicity (Cantoni et al., 1989). In G86R mutant SOD1transgenic mice, no significant decrease in the number of calbindin-positive neurons was observed and calbindin-containing neurons in the anterior horn were protected (Morrison et al., 1996). Moreover, no significant decrease in the number of calbindin D-28k immunoreactive neurons in G93A SOD1transgenic mice was observed (Knirsch et al., 2001). Extraocular motoneurons from mutant SOD1-transgenic mice, which express abundant levels of calbindin D-28k and/or parvalbumin, are less likely to degenerate as motor neuron disease develops (Nimchinsky et al., 2000; Siklos et al., 1999). Furthermore, oculomotor neurons which possess abundant parvalbumin in vivo have five- to sixfold larger calciumbuffering capacities than those of hypoglossal and spinal motoneurons (Vanselow and Keller, 2000) and specialized mechanisms to maintain calcium homeostasis as compared with vulnerable spinal and hypoglossal motoneurons (Siklos et al., 1999). Transgenic mice overexpressing parvalbumin in spinal motoneurons interbred with mutant SOD1 have significantly reduced motoneuron loss and have delayed disease onset and prolonged survival when compared with
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mice with only the mutant SOD1 transgene (Beers et al., 2001). Thus, the inability to handle an increased calcium load, related to low levels of calbindin D-28k and/or parvalbumin, may contribute to selective vulnerability of motoneurons in ALS-linked mutant SOD1-transgenic mice. To our knowledge, this is the first report of parvalbumin immunoreactivity in G93A mutant SOD1-transgenic mice. The diffuse and severe loss of parvalbumin immunoreactivity of large motor neurons even at presymptomatic stages, the absence of calbindin D-28k immunoreactivity of normal large motor neurons and high expression of calbindin D-28k and/or parvalbumin in extraocular motoneurons suggest that these calcium-binding proteins may contribute to selective vulnerability and an early loss of function of large motor neurons, probably leading to the degeneration of anterior horn neurons in this SOD1-transgenic mouse model. Motor neurons expressing mutant SOD1 are more susceptible to glutamate-mediated cell death than are wild-type neurons (Roy et al., 1998), but the susceptibility of motor neurons can be reversed by coexpression of the calbindin or overexpression of parvalbumin because changes in Ca2+ homeostasis are thought to participate in the degeneration of neurons in ALS patients (Julien, 2001) and AMPA/kainate-induced Ca2+-dependent cell death in vitro (Bosch et al., 2002). Recently, overexpression of the calcium-binding protein, calbindin D-28k, was reported as protective against mutant-SOD1-mediated death of PC12 cells (Couillard-Despres et al., 1998). Thus, new therapeutic strategies may be developed using agents which selectively modulate specific deficiencies in calcium-binding proteins.
4.
Experimental procedures
4.1.
Experimental animals and clinical assessment
Transgenic mice expressing G93A mutant human SOD1 were used in this study (Gurney et al., 1994). The mice were originally obtained from the Jackson Laboratory (B6SJL-TgN (SOD1-G93A) 1 Gurdl, Bar Harbor, ME, USA) and were backcrossed to a C57BL/6 background by mating hemizygote males with inbred C57BL/6 female mice (C57BL/6CrSlc, Nihon SLC, Shizuoka, Japan), thus producing transgenic (Tg) and nontransgenic (non-Tg) littermates. The transgenic progeny was identified by polymerase chain reaction (PCR) amplification of tail DNA with specific primers for exon 4 (Rosen et al., 1993). This G93A SOD1 mutant mouse expressed a relatively low mutant protein (gene copy 10). At around 32 weeks of age, the G93A transgenic mice developed progressive muscle weakness and spasticity in one or more limbs beginning with a posterior limb. One to two weeks later, they could not feed themselves due to severe paralysis expressed by the hyperextension of their hindlimbs. The G93A Tg and non-Tg mice were examined simultaneously. The animals were divided into four groups: early presymptomatic Tg (aged 24 weeks, n = 2), late presymptomatic Tg (aged 28 weeks, n = 2), early symptomatic Tg (aged 32 weeks, n = 2) and end-stage Tg mice (aged 35 weeks, n = 1). Throughout the present study, the mice were treated in accordance with the declaration of Helsinki and the guiding principles for the care and use of laboratory animals.
4.2.
201
Histopathological analysis
Seven transgenic and seven non-transgenic wild-type mice were sacrificed at ages ranging from 24 to 35 weeks. All mice were deeply anesthetized with ether and perfused intracardially with heparinized saline (pH 7.4) followed by perfusion with ice-cold 4% paraformaldehyde (Katayama Chemical, Osaka, Japan) in 0.1 M phosphate buffer (pH 7.4). The brain stems and the spinal cords were rapidly removed and postfixed by immersion in the same fixative (5 days, 4 °C). Crosssections of the spinal cord and serial cross-sections of the brain stem were embedded in paraffin, sectioned (4 μm) and stained with hematoxylin and eosin and Nissl stains.
4.3.
Immunohistochemistry
We employed a monoclonal parvalbumin (PARV-19) (Sigma, Saint Louis, Missouri; diluted 1:2000), a monoclonal calbindin D-28k (Sigma, Saint Louis, Missouri; diluted 1:400), a monoclonal anti-glial fibrillary acidic protein (GFAP) antibody (Dako; diluted 1:400) and a mouse monoclonal antibody to rat phosphorylated neurofilament antibody (SMI-31, Sternberger Monoclonals; diluted 1:10,000). Sections (4 μm thick) of the paraffin-embedded brain stems and spinal cords were deparaffinized, treated with nonimmune serum as the blocking reagent, quenched with 3% H2O2 and incubated overnight at 4 °C with the primary antibodies. Antibody binding was visualized using the avidin–biotin– immunoperoxidase complex (ABC) method employing an Elite ABC kit (Vector Laboratories, Burlingame, CA) following the manufacturer's recommendations. 3, 3′-Diaminobenzidine tetrahydrochloride (DAB) was the final chromogen. Selected sections were exposed to primary antiserum that had been preabsorbed with excess amounts of parvalbumin and calbindin D-28k following the manufacturer's recommendations (diluted at 1:5000, and 1 μg of the recombinant protein was added to 1 ml of the diluted antibody solution; SWant, Bellinzona, Switzerland). Sections from which the primary antibody was omitted served as negative reaction controls.
4.4.
Immunoblotting analysis
After the mice (aged 36 weeks) were deeply anesthetized, the lumbar cords of decapitated animals were obtained. The anterior half of each sample, including anterior horn, was removed and frozen in powdered dry ice. Tissue samples were then homogenized in lysis buffer (20 mM Tris–HCl, 10% sucrose, 10 mM benzamidine, 1 mM EDTA, 5 mM EGTA, 20 μg/ml aprotinin, leupeptin, antipain and pepstatin A and 1 mM PMSF). The homogenates were then centrifuged at 3000×g for 10 min at 4 °C, and the supernatants were collected. For the immunoblotting study, sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to our previous report (Hayashi et al., 1997). The blots were developed with SuperSignal West Dura Extended Duration Substrate (PIERCE, 34075, Rockford, IL). Images of the blots were captured with a luminoimage analyzer (LAS 1000-mini, Fuji film, Tokyo), and densitometric analysis was performed using Scion image.
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Acknowledgments This work was supported by a Grant-in-Aid for General Scientific Research (C) from the Japanese Ministry of Education, Science and Culture, and a grant from the Japan ALS Association.
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Rahmani, Z., Krizus, A., Mckenna-Yasek, D., Cayabyab, A., Gaston, S.M., Berger, R., Tanzi, R.E., Halperin, J.J., Herzfeldt, B., Van den Bergh, R., Hung, W.-Y., Bird, T., Deng, G., Mulder, D.W., Smyth, C., Laing, N.G., Soriano, E., Pericak-Vance, M.A., Haines, J., Rouleau, G.A., Gusella, J.S., Horvitz, H.R., Brown Jr., R.H., 1993. Mutation in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362, 59–62. Rothstein, J.D., Jin, L., Dykes-Hoberg, M., Kuncl, R.W., 1993. Chronic inhibition of glutamate uptake produces a model of slow neurotoxicity. Proc. Natl. Acad. Sci. U. S. A. 90, 6591–6595. Roy, J., Minotti, S., Dong, L., Figlewicz, D.A., Durham, H.D., 1998. Glutamate potentiates the toxicity of mutant Cu/Znsuperoxide dismutase in motor neurons by postsynaptic calcium-dependent mechanisms. J. Neurosci. 18, 9673–9684. Shaw, P.J., 1994. Excitotoxicity and motor neurone disease: a review of the evidence. J. Neurol. Sci. 124, 6–13 (Suppl.). Shaw, P.J., Eggett, C.J., 2000. Molecular factors underlying selective vulnerability of motor neurons to neurodegeneration in amyotrophic lateral sclerosis. J. Neurol. 247, 117–127.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Parvalbumin and calbindin D-28k immunoreactivity in transgenic mice with a G93A mutant SOD1 gene Shoichi Sasaki a,⁎, Hitoshi Warita b , Takashi Komori c , Tetsuro Murakami d , Koji Abe d , Makoto Iwata a a
Department of Neurology, Neurological Institute, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan Department of Neurology, Yonezawa National Hospital, Japan c Department of Clinical Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Japan d Department of Neurology, Okayama University Medical School, Japan b
A R T I C LE I N FO
AB S T R A C T
Article history:
Immunohistochemical study was performed to examine if calcium-binding proteins are
Accepted 28 January 2006
involved in the degeneration of motor neurons in the brain stems and the spinal cords of
Available online 20 March 2006
transgenic mice carrying a G93A mutant human SOD1 gene. Specimens from age-matched non-transgenic wild-type mice served as controls. In the spinal cord of the controls, the
Keywords:
density of parvalbumin-immunoreactive neurons was highest in the large anterior horn
Amyotrophic lateral sclerosis
neurons and lower in the posterior horn neurons in the spinal cord. On the other hand,
Transgenic mice
calbindin D-28k immunoreactivity was much less apparent than that observed with
SOD1 mutation
parvalbumin antisera. Rexed's lamina II was densely immunostained for calbindin D-28k,
Parvalbumin
whereas, in the anterior horn, calbindin-D-28k-positive small neurons were barely
Calbindin D-28k
dispersed in a scattered pattern. In transgenic mice, parvalbumin-positive anterior horn
Immunohistochemistry
neurons were severely reduced, even at the presymptomatic stage, whereas calbindinpositive neurons were largely preserved. At the symptomatic stage, both parvalbumin and calbindin D-28k immunoreactivity markedly diminished or disappeared in the anterior horn. Immunoblotting analysis revealed a significant reduction of immunoreactivity to parvalbumin antibody in transgenic mice compared with the controls. In the brain stem, parvalbumin-positive oculomotor and abducens neurons and the calbindin D-28k-positive sixth nucleus were well-preserved in transgenic mice as well as in the controls. Thus, the diffuse and severe loss of parvalbumin immunoreactivity of large motor neurons even at early stages in SOD1-transgenic mice and the absence of calbindin D-28k immunoreactivity of normal large motor neurons suggest that these calcium-binding proteins may contribute to selective vulnerability and an early loss of function of large motor neurons in this SOD1transgenic mouse model. © 2006 Elsevier B.V. All rights reserved.
⁎ Corresponding author. Fax: +81 3 5269 7324. E-mail address: [email protected] (S. Sasaki). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.01.129
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1.
197
Introduction
Parvalbumin and calbindin D-28k are calcium-binding proteins that appear to be involved in the buffering of free intracellular calcium, and they play an important role in regulating neuronal calcium homeostasis (Baimbridge et al., 1992; Heizman and Braun, 1992). It has been suggested that the selective neuronal distribution of calcium-binding proteins may be a determinant of vulnerability to excitotoxicity, in which a disturbance of intracellular calcium homeostasis plays a major role (Shaw, 1994). A number of studies suggested that at least two factors are considered to contribute to selective vulnerability of motor neurons: the high density of Ca-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type of glutamate receptors (Carriedo et al., 1996; Ikonomidou et al., 1996; Rothstein et al., 1993) and the low expression of the calcium-binding proteins parvalbumin and/or calbindin D-28k in motor neurons (Celio, 1990; Ince et al., 1993). However, little is known about immunoreactivity of calcium-binding proteins in mutant SOD1-transgenic mice (Knirsch et al., 2001; Morrison et al., 1996; Nimchinsky et al., 2000; Siklos et al., 1999), especially parvalbumin immunoreactivity in G93A mutant SOD1-transgenic mice. In this study, we performed immunohistochemical investigation in order to determine whether or not these calcium-binding proteins are involved in the degeneration of motor neurons in transgenic mice carrying a G93A mutant human SOD1 gene from presymptomatic to symptomatic stages.
Fig. 1 – Many anterior horn neurons are immunostained for parvalbumin (control case, 24 weeks, lumbar spinal cord). The somata of anterior horn neurons and neuronal processes are immunoreactive.
2.
Results
2.2.
2.1.
Non-transgenic littermates
At the age of 24 weeks (early presymptomatic), all levels of the spinal cord were free from neuronal depletion in the anterior horns. Parvalbumin immunoreactivity in the anterior horns was diminished at the lumbar level (Fig. 4) but was well-preserved at the cervical level. There was no difference in calbindin D-28k immunoreactivity between controls and transgenic mice. At the age of 28 weeks (late presymptomatic), slight neuronal loss of anterior horn cells (those exceeding 35 μm in the mean of long and short diameters) was recognized at the cervical (28.0 ± 3.4) and lumbar (27.6 ± 5.2) levels, showing significantly lower values than those of the controls (36.1 ± 2.9, 35.4 ± 2.8, respectively) (P < 0.01). There were prominent vacuolar changes of various sizes in the neuropil of the anterior horns and anterior roots in the anterior column. Lewy-body-like inclusions were occasionally observed in the neuropil of the anterior horns. Parvalbumin-positive anterior horn neurons were much more reduced at all levels of the spinal cord, as compared with the early presymptomatic stage, and parvalbumin-positive posterior horn neurons also decreased in number, though to a lesser extent. There was no significant reduction in the number of calbindin D-28k immunoreactive neurons. At the age of 32 weeks (early symptomatic stage), the anterior horns showed a moderate neuronal loss of anterior horn cells at the cervical (21.8 ± 11.7) and lumbar (25.4 ± 4.6) levels; these values were significantly lower than those obtained with the controls (37.4 ± 2.2, 32.9 ± 5.2, respectively) (P < 0.01). Vacuolar
Parvalbumin immunostaining in the spinal cord was much more intense than that observed with calbindin D-28k antisera. Both the gray and white matter of all segments of the spinal cord showed parvalbumin immunoreactivity. In the gray matter, the perikarya and neuronal processes of many small to large anterior horn neurons, and of neurons of intermediate gray (lamina VII), and, to a lesser extent, of neurons in the posterior horn (Rexed's laminae II–VI), were positively immunostained for parvalbumin (Fig. 1). The density of parvalbumin-immunoreactive neurons was highest in the large anterior horn cells. Some neurons around the central canal (lamina X) and in Clarke's nucleus also exhibited positive immunoreactivity for parvalbumin. In the white matter, axons of all funiculi were diffusely immunostained. Parvalbumin-positive axons were regularly seen in the ventral root, as well as in the dorsal root. On the other hand, many small cells, together with their processes, in the posterior horn (Rexed's lamina II) were consistently immunoreactive with anti-calbindin D-28k antibody (Fig. 2). Calbindin-positive medium-sized or small neurons were less numerous and barely dispersed in a scattered pattern through the ventral horn; however, the large anterior horn cells were not immunoreactive (Fig. 3). Due to the low number of these neurons, it was difficult to assess the quantitative changes in calbindin D-28k immunoreactive neurons. All funiculi of the
white matter were not, or only weakly, immunostained for calbindin D-28k. The ventral root and the dorsal root were not immunostained. The blood vessels were not immunostained for parvalbumin or calbindin D-28k. Somata of the sixth and seventh cranial nerve nuclei were positively immunostained for calbindin D-28k, whereas perikarya and fibers of the third nucleus exhibited no immunoreactivity. On the other hand, oculomotor (the nucleus of Darkschewitsch), abducens and facial neurons were parvalbumin-positive.
Transgenic mice
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analysis of the band using Scion image revealed a significant reduction (31%) of immunoreactivity to parvalbumin antibody in transgenic mice compared with the controls (Student's t test, P < 0.01) (Fig. 8). On the other hand, there was no detectable expression of calbindin D-28k protein in the spinal cord of either the transgenic or the non-transgenic group.
3.
Fig. 2 – Many small cells in the posterior horn (Rexed's lamina II) are immunostained for calbindin D-28k (control case, 28 weeks, cervical spinal cord).
changes were observed in the neuropil of the anterior horns and the anterior roots, and Lewy-body-like inclusions were frequently demonstrated within the neuronal processes including the proximal axons. Parvalbumin immunoreactivity was markedly reduced in the anterior horns. Calbindin D-28k immunoreactivity was not observed in the anterior horns, whereas no differences were observed as regards the distribution and density of calbindin D-28k in the posterior horns of transgenic mice and the controls. At the age of 35 weeks (end-stage), severe neuronal loss of the anterior horn neurons accompanied by prominent astrogliosis was observed at the cervical (13.0 ± 4.6) and lumbar (7.0 ± 0.9) levels, showing significantly lower values than those observed in the controls (31.5 ± 6.8, 32.8 ± 2.5, respectively) (P < 0.001). Lewybody-like inclusions were frequently observed in the neuronal processes. However, vacuolar changes were less prominent in the anterior horns. Parvalbumin immunoreactivity was not shown in the anterior horns (Fig. 5) nor was calbindin D-28k-positive immunoreactivity seen in the anterior horns, while in the posterior horn (Rexed's lamina II), calbindin D28k immunoreactivity was well-preserved, as was observed in the controls. The immunoreactivity of parvalbumin and calbindin D-28k in the white matter was well-preserved, but it did decrease in the ventral root as well as in the dorsal root. The blood vessels were not immunostained for either protein. Neither the sections processed with omission of the primary antibodies nor those incubated with the preabsorbed antibodies at the late presymptomatic stage (28 weeks) showed any immunoreaction product deposits (Fig. 6). In the brain stem, calbindin D-28k immunoreactivity of the seventh nucleus was decreased as compared with the controls, whereas it was well-preserved in the sixth nucleus. Parvalbumin immunoreactivity was well-preserved in oculomotor (Fig. 7) and abducens neurons, although it was decreased in facial neurons.
2.3.
Discussion
In non-transgenic littermates of the controls, both parvalbumin and calbindin D-28k were specific for neurons and their processes and were not observed in the glial and supporting cells. The density of parvalbumin-immunoreactive neurons was highest in the large anterior horn neurons and lower in the posterior horn cells, whereas calbindin D-28k was predominantly expressed in the small anterior neurons (presumably interneurons) and the posterior horn (Rexed's lamina II). The distribution of parvalbumin and calbindin D28k immunoreactivity in the mouse spinal cords in this study was almost the same as that observed in rat spinal cords (Celio, 1990; Laslo et al., 2000; Lim et al., 2000; Ren and Ruda, 1994), although Zhang et al. reported parvalbumin and calbindin D-28k immunoreactivity in a few medium-sized (about 30–35 μm in diameter) and small (about 15–18 μm in diameter) neurons in the ventral horn, calbindin D-28kpositive neurons in the superficial layers of the posterior horn and parvalbumin-positive neurons in layers II–III of the dorsal horn of the rat spinal cord (Zhang et al., 1990). On the other hand, in the human spinal cord, parvalbumin is confined to a few interneuronal processes in the ventral horn only, while calbindin is present in many interneurons
Immunoblotting
Quantitative Western blot analysis of extracted mice lumbar spinal cord protein for parvalbumin showed a band corresponding to parvalbumin protein (12 kDa) in both the transgenic and non-transgenic groups. Then, quantitative
Fig. 3 – Calbindin-positive small-sized neurons are dispersed in a scattered pattern in the anterior horn, but large anterior horn cells are not immunostained (control case, 24 weeks, lumbar spinal cord).
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199
Fig. 4 – (A) At the age of 24 weeks (early presymptomatic stage), parvalbumin immunoreactivity is diminished in the anterior horn in the lumbar spinal cord. (B) Age-matched control case for comparison.
and fibers within the gray matter (Ince et al., 1993). However, lower motor neurons at all spinal levels are negatively immunostained for both antibodies (Ince et al., 1993). Thus, the distribution of immunoreactivity of these calcium-binding proteins in the mice or rat spinal cords is different from that of the human (Celio, 1990), as was confirmed by this study. The functions of parvalbumin and calbindin are not still clear, but a number of reports suggest that these proteins may protect cells by binding and buffering increased intracellular
calcium (Chard et al., 1993; Heizman and Braun, 1992; Lledo et al., 1992; Mattson et al., 1991; Miller, 1991). Selective vulnerability of motoneurons to an increased calcium load may be related to their low expression of the calcium-binding proteins calbindin D-28k and/or parvalbumin (Celio, 1990). Excitotoxic processes are involved in motoneuron degeneration, whereby overstimulation by glutamate results in elevated levels of intracellular calcium (Shaw and Eggett, 2000), which in turn initiates a cascade of destructive metabolic processes such as
Fig. 5 – (A) At the age of 35 weeks (end-stage), almost no parvalbumin immunoreactivity in the anterior horn can be seen (lumbar spinal cord). (B) Age-matched control case for comparison.
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Fig. 8 – Western blot analysis shows a band corresponding to parvalbumin (12 kDa) in both transgenic and non-transgenic groups, and immunoreactivity to parvalbumin antibody in transgenic mice was significantly reduced by 31% compared with the controls (Student's t test, P < 0.01).
Fig. 6 – The section incubated with the preabsorbed antibody shows that the parvalbumin immunostaining was completely prevented (lumbar spinal cord). the activation of Ca2+-dependent protein kinases, endonucleases and phospholipases (Choi, 1988; Nicotera and Orrenius, 1992). Increased intracellular calcium has been shown to accelerate the production of nitric oxide and superoxide (Coyle and Puttfarcken, 1993; Dawson et al., 1994; Lafon-Cazal et al., 1993), free radicals that are precursors to both peroxynitrite (Beckman and Crow, 1993) and hydrogen peroxide (Fridovich, 1974). In amyotrophic lateral sclerosis (ALS), calcium is increased within neurons and motor nerve terminals of biopsied human muscle specimens, as well as in motor nerve terminals of mice following passive transfer of ALS immunoglobulin G (IgG) (Krieger et al., 1994; Pullen and Humphreys, 2000; Siklos et al., 1996). Calcium is also increased in vesicular structures within spinal motoneurons of trans-
Fig. 7 – At the end-stage, parvalbumin immunoreactivity in the accessory oculomotor nucleus (the nucleus of Darkschewitsch) is well-preserved. Scale bars, Figs. 1–3, 7): 50 μm, Figs. 4, 5: 125 μm, Fig. 6: 200 μm.
genic mice expressing mutant human SOD1 (Siklos et al., 1998). In human autopsy specimens, motoneurons relatively deficient in the calcium-binding proteins (e.g. spinal and hypoglossal motoneurons) are lost early in ALS, whereas motoneurons expressing high levels of these proteins (cranial nerves III, IV, VI and Onuf's nucleus motoneurons) are relatively spared (Alexianu et al., 1994; Elliot and Snider, 1995; Ince et al., 1993) that is true in mutant SOD1-transgenic mice in this study. These results suggest that cells containing these calcium-buffering proteins may be less vulnerable to the pathological process associated with motor neuron disease and that calcium-binding proteins play a role in protecting neurons from degeneration in ALS. However, the role of calcium-binding proteins in establishing this differential vulnerability of motoneurons to degeneration has been questioned by recent observations that vulnerable spinal motoneurons express parvalbumin (Philippe et al., 1993), which is consistent with the results of the present study. A recent demonstration of reduced SOD1 activity may implicate motoneuron damage through changes in motoneuron calcium homeostasis since intracellular calcium chelators were shown to prevent free-radical-induced DNA breakdown and cytotoxicity (Cantoni et al., 1989). In G86R mutant SOD1transgenic mice, no significant decrease in the number of calbindin-positive neurons was observed and calbindin-containing neurons in the anterior horn were protected (Morrison et al., 1996). Moreover, no significant decrease in the number of calbindin D-28k immunoreactive neurons in G93A SOD1transgenic mice was observed (Knirsch et al., 2001). Extraocular motoneurons from mutant SOD1-transgenic mice, which express abundant levels of calbindin D-28k and/or parvalbumin, are less likely to degenerate as motor neuron disease develops (Nimchinsky et al., 2000; Siklos et al., 1999). Furthermore, oculomotor neurons which possess abundant parvalbumin in vivo have five- to sixfold larger calciumbuffering capacities than those of hypoglossal and spinal motoneurons (Vanselow and Keller, 2000) and specialized mechanisms to maintain calcium homeostasis as compared with vulnerable spinal and hypoglossal motoneurons (Siklos et al., 1999). Transgenic mice overexpressing parvalbumin in spinal motoneurons interbred with mutant SOD1 have significantly reduced motoneuron loss and have delayed disease onset and prolonged survival when compared with
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mice with only the mutant SOD1 transgene (Beers et al., 2001). Thus, the inability to handle an increased calcium load, related to low levels of calbindin D-28k and/or parvalbumin, may contribute to selective vulnerability of motoneurons in ALS-linked mutant SOD1-transgenic mice. To our knowledge, this is the first report of parvalbumin immunoreactivity in G93A mutant SOD1-transgenic mice. The diffuse and severe loss of parvalbumin immunoreactivity of large motor neurons even at presymptomatic stages, the absence of calbindin D-28k immunoreactivity of normal large motor neurons and high expression of calbindin D-28k and/or parvalbumin in extraocular motoneurons suggest that these calcium-binding proteins may contribute to selective vulnerability and an early loss of function of large motor neurons, probably leading to the degeneration of anterior horn neurons in this SOD1-transgenic mouse model. Motor neurons expressing mutant SOD1 are more susceptible to glutamate-mediated cell death than are wild-type neurons (Roy et al., 1998), but the susceptibility of motor neurons can be reversed by coexpression of the calbindin or overexpression of parvalbumin because changes in Ca2+ homeostasis are thought to participate in the degeneration of neurons in ALS patients (Julien, 2001) and AMPA/kainate-induced Ca2+-dependent cell death in vitro (Bosch et al., 2002). Recently, overexpression of the calcium-binding protein, calbindin D-28k, was reported as protective against mutant-SOD1-mediated death of PC12 cells (Couillard-Despres et al., 1998). Thus, new therapeutic strategies may be developed using agents which selectively modulate specific deficiencies in calcium-binding proteins.
4.
Experimental procedures
4.1.
Experimental animals and clinical assessment
Transgenic mice expressing G93A mutant human SOD1 were used in this study (Gurney et al., 1994). The mice were originally obtained from the Jackson Laboratory (B6SJL-TgN (SOD1-G93A) 1 Gurdl, Bar Harbor, ME, USA) and were backcrossed to a C57BL/6 background by mating hemizygote males with inbred C57BL/6 female mice (C57BL/6CrSlc, Nihon SLC, Shizuoka, Japan), thus producing transgenic (Tg) and nontransgenic (non-Tg) littermates. The transgenic progeny was identified by polymerase chain reaction (PCR) amplification of tail DNA with specific primers for exon 4 (Rosen et al., 1993). This G93A SOD1 mutant mouse expressed a relatively low mutant protein (gene copy 10). At around 32 weeks of age, the G93A transgenic mice developed progressive muscle weakness and spasticity in one or more limbs beginning with a posterior limb. One to two weeks later, they could not feed themselves due to severe paralysis expressed by the hyperextension of their hindlimbs. The G93A Tg and non-Tg mice were examined simultaneously. The animals were divided into four groups: early presymptomatic Tg (aged 24 weeks, n = 2), late presymptomatic Tg (aged 28 weeks, n = 2), early symptomatic Tg (aged 32 weeks, n = 2) and end-stage Tg mice (aged 35 weeks, n = 1). Throughout the present study, the mice were treated in accordance with the declaration of Helsinki and the guiding principles for the care and use of laboratory animals.
4.2.
201
Histopathological analysis
Seven transgenic and seven non-transgenic wild-type mice were sacrificed at ages ranging from 24 to 35 weeks. All mice were deeply anesthetized with ether and perfused intracardially with heparinized saline (pH 7.4) followed by perfusion with ice-cold 4% paraformaldehyde (Katayama Chemical, Osaka, Japan) in 0.1 M phosphate buffer (pH 7.4). The brain stems and the spinal cords were rapidly removed and postfixed by immersion in the same fixative (5 days, 4 °C). Crosssections of the spinal cord and serial cross-sections of the brain stem were embedded in paraffin, sectioned (4 μm) and stained with hematoxylin and eosin and Nissl stains.
4.3.
Immunohistochemistry
We employed a monoclonal parvalbumin (PARV-19) (Sigma, Saint Louis, Missouri; diluted 1:2000), a monoclonal calbindin D-28k (Sigma, Saint Louis, Missouri; diluted 1:400), a monoclonal anti-glial fibrillary acidic protein (GFAP) antibody (Dako; diluted 1:400) and a mouse monoclonal antibody to rat phosphorylated neurofilament antibody (SMI-31, Sternberger Monoclonals; diluted 1:10,000). Sections (4 μm thick) of the paraffin-embedded brain stems and spinal cords were deparaffinized, treated with nonimmune serum as the blocking reagent, quenched with 3% H2O2 and incubated overnight at 4 °C with the primary antibodies. Antibody binding was visualized using the avidin–biotin– immunoperoxidase complex (ABC) method employing an Elite ABC kit (Vector Laboratories, Burlingame, CA) following the manufacturer's recommendations. 3, 3′-Diaminobenzidine tetrahydrochloride (DAB) was the final chromogen. Selected sections were exposed to primary antiserum that had been preabsorbed with excess amounts of parvalbumin and calbindin D-28k following the manufacturer's recommendations (diluted at 1:5000, and 1 μg of the recombinant protein was added to 1 ml of the diluted antibody solution; SWant, Bellinzona, Switzerland). Sections from which the primary antibody was omitted served as negative reaction controls.
4.4.
Immunoblotting analysis
After the mice (aged 36 weeks) were deeply anesthetized, the lumbar cords of decapitated animals were obtained. The anterior half of each sample, including anterior horn, was removed and frozen in powdered dry ice. Tissue samples were then homogenized in lysis buffer (20 mM Tris–HCl, 10% sucrose, 10 mM benzamidine, 1 mM EDTA, 5 mM EGTA, 20 μg/ml aprotinin, leupeptin, antipain and pepstatin A and 1 mM PMSF). The homogenates were then centrifuged at 3000×g for 10 min at 4 °C, and the supernatants were collected. For the immunoblotting study, sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to our previous report (Hayashi et al., 1997). The blots were developed with SuperSignal West Dura Extended Duration Substrate (PIERCE, 34075, Rockford, IL). Images of the blots were captured with a luminoimage analyzer (LAS 1000-mini, Fuji film, Tokyo), and densitometric analysis was performed using Scion image.
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Acknowledgments This work was supported by a Grant-in-Aid for General Scientific Research (C) from the Japanese Ministry of Education, Science and Culture, and a grant from the Japan ALS Association.
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