byThe Jackson Laboratory (Bar Harbor, ME) under the strain ... 130(3). -. +. +/-. 199(2). +/-. ++. +/-. -. Number in parentheses represents the animal number used.
Proc. Natl. Acad. Sci. USA Vol. 93, pp. 3155-3160, April 1996
Neurobiology
Transgenic mice carrying a human mutant superoxide dismutase transgene develop neuronal cytoskeletal pathology resembling human amyotrophic lateral sclerosis lesions (free radicals/neurofilaments/spheroids/motor
neuron
disease)
PANG-HSIEN TU*, PRAMOD RAJU*, KATHRYN A. ROBINSON*, MARK E. GURNEYt, JOHN Q. TROJANOWSKI*, LEE*t
AND VIRGINIA M.-Y.
*Department of Pathology and Laboratory Medicine, Division of Anatomic Pathology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4283; and tDepartment of Cell, Molecular, and Structural Biology, Northwestern University Medical School, Chicago, IL 60611
Communicated by Britton Chance, Johnson Research Foundation, Philadelphia, PA, December 19, 1995 (received for review April 6, 1995)
neuronal intermediate filaments (IFs) (7, 8). Although the of ALS remains elusive, the presence of similar pathology in most ALS cases (9) suggests that common pathogenetic mechanisms may lead to the development of this neurodegenerative disease. A recent breakthrough in ALS was the discovery of a variety of mutations in the human Cu,Zn superoxide dismutase gene (SOD1) in "20% of FALS cases (10) that cause structural defects in superoxide dismutase (SOD) (11). Further, FALS patients exhibit compromised SOD activity (12). Thus, it has been hypothesized that a decrease in SOD activity leads to the accumulation of highly reactive hydroxyl radical and peroxynitrite, which eventually results in the death of motor neurons and the ALS phenotype. However, transgenic mice carrying a human SOD1 transgene with the Gly-93 -> Ala mutation (G1H mice) showed an ALS-like phenotype, even though they evidenced a 4-fold increase in SOD enzymatic activity (13). Thus, these data implied that the ALS phenotype was due to a gain-of-function in the human SOD1 mutant protein (13, 14), and recent studies suggest that mutant SOD1 may promote apoptosis (15). Previous studies have shown that the spinal cords of the G1H mice developed pathological abnormalities characterized by prominent vacuolar degeneration originating from dilations of the endoplasmic reticulum and mitochondria at the early stages of the disease and marked neuronal loss with hyaline filamentous inclusions in some of the surviving neurons at the terminal stages of the disease (16, 17). To gain further insights into the pathogenesis of FALS, the studies described in this paper were designed to characterize alterations in the neuronal cytoskeleton in the G1H mice and to compare these alterations with those seen in human ALS.
Mutations in the human Cu,Zn superoxide ABSTRACT dismutase gene (SOD1) are found in 20% of kindreds with familial amyotrophic lateral sclerosis. Transgenic mice (line G1H) expressing a human SOD1 containing a mutation of Gly-93 -> Ala (G93A) develop a motor neuron disease similar to familial amyotrophic lateral sclerosis, but transgenic mice (line N1029) expressing a wild-type human SOD1 transgene do not. Because neurofilament (NF)-rich inclusions in spinal motor neurons are characteristic of amyotrophic lateral sclerosis, we asked whether mutant G1H and/or N1029 mice develop similar NF lesions. NF inclusions (i.e., spheroids, Lewy body-like inclusions) were first detected in spinal cord motor neurons of the G1H mice at 82 days of age about the time these mice first showed clinical evidence of disease. Other neuronal intermediate filament proteins (a-internexin, peripherin) also accumulated in these spheroids. The onset of accumulations of ubiquitin immunoreactivity in the G1H mice paralleled the emergence ofvacuoles and NF-rich spheroids in neurons, but they did not colocalize exclusively with spheroids. In contrast, NF inclusions were not seen in the N1029 mice until they were 132 days old, and ubiquitin immunoreactivity was not increased in the N1029 mice even at 199 days of age. Astrocytosis in spinal cord was associated with a marked increase in glial fibrillary acidic protein immunoreactivity in the G1H mice, but not in the N1029 mice. Finally, comparative studies revealed a striking similarity between the cytoskeletal pathology in the G1H transgenic mice and in patients with amyotrophic lateral sclerosis. These findings link a specific SOD1 mutation with alterations in the neuronal cytoskeleton of patients with amyotrophic lateral sclerosis. Thus, neuronal cytoskeletal abnormalities may be implicated in the pathogenesis of human familial amyotrophic lateral sclerosis.
cause
MATERIALS AND METHODS
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that mainly affects motor neurons in cortex, brain stem, and spinal cord (1). The diagnosis of ALS is based on a constellation of clinical and electromyographic abnormalities of the motor system (2, 3). For example, ALS typically starts with asymmetric weakness in one or more limbs, and it eventually progresses to all limbs, leading to complete paralysis (4). ALS occurs both in sporadic (SALS) and familial (FALS) forms (5, 6). FALS constitutes -10% of all ALS cases, and FALS is mainly inherited in an autosomal dominant fashion. Despite clinical and genetic heterogeneity among ALS cases, patients afflicted with this disease develop a similar pathology characterized by a selective loss of motor neurons and the accumulation of spheroids in proximal axons of the surviving motor neurons. These spheroids are composed primarily of
Generation of Human SOD1 Transgenic Mice. The G1H line of transgenic mice carry a human SOD1 gene with a Gly-93 -> Ala substitution (G93A). These derive from a described Gl line (13) and have a 40% expansion in transgene copy number (P. Zhai and M.E.G., unpublished data). The G1H mice have been deposited with the Induced Mutant Resource maintained by The Jackson Laboratory (Bar Harbor, ME) under the strain designation C57BL/6J-TgN(SOD1-G93A)lGur. The N1029 Abbreviations: ALS, amyotrophic lateral sclerosis; FALS, familial ALS; NF, neurofilament; IF, intermediate filament; NFL, NFM, and NFH, low-molecular weight, middle-molecular-weight and highmolecular-weight, respectively; GFAP, glial fibrillary acidic protein; SALS, sporadic ALS; mAb, monoclonal antibody. tTo whom reprint requests should be addressed at: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Hospital of University of Pennsylvania, Room A009, Maloney Basement, 3400 Spruce Street, Philadelphia, PA
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
19104.
3155
3156
Proc. Natl. Acad. Sci. USA 93 (1996)
Neurobiology: Tu et al.
Table 1. Summary of pathology in SOD1 transgenic mice Age Vacuolation Spheroid* Ubiquitination Astrocytosis G1H 45 (2)
82(2) 111 (2) 130 (3) 140 (3)
N1029 51 (2) 70 (2) 98 (2)
130(3) 199(2)
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Number in parentheses represents the animal number used. -, Absence of vacuolation, spheroids, ubiquitination or astrocytosis; +/-, little or no vacuolation or ubiquitination. *+, 4 times more Cu,Zn SOD enzymatic activity than wild-type, nontransgenic mice (13). The earliest signs of clinical disease in G1H mice (hyperreflexia, crossed spread of spinal reflexes, and shaking of the limbs when suspended in the air) occur by 91 ± 14 days of age; this develops into a progressively worsening paresis involving primarily the hind Table 2. List of antibodies for immunohistochemistry
limbs with atrophy of the skeletal musculature. At end-stage disease, which occurs in the G1H mice at 136 ± 7 days, the mice are severely paralyzed and unable to forage for food or water. The N1029 mice do not show any obvious clinical phenotype for up to 400 days (M.E.G., unpublished work). The G1H mice used in this study ranged from 45 to 144 days of age, and the N1029 mice ranged from 51 to 199 days of age. At least two animals were sacrificed and examined at each age group (Table 1). Immunohistochemistry. All animals were lethally anesthetized and perfusion-fixed with either 70% ethanol/150 mM NaCl or freshly prepared 4% paraformaldehyde/phosphatebuffered saline (pH 7.4). Tissues remained in the same fixative overnight before further dissection. Both human and mouse tissue samples were processed for immunohistochemistry as described (18). The clinical and pathological features of the two human ALS specimens used in this study were reported earlier (18). The antibodies to neurofilament (NF) subunits, other neuronal IF proteins, and selected additional polypeptides studied here have been characterized previously, and the properties of these antibodies are summarized in Table 2. Quantitation was done by counting the number of NF inclusions per section after staining with anti-NF antibodies. Sections with 10 or more NF aggregates per section were given a + + score, whereas sections with 1-10 inclusions per section were scored as +. Spinal cord sections without any aggregates were scored as - (see Table 1). Each section was evaluated independently by two observers, and 50 sections including all levels of the spinal cord were scored per animal.
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FIG. 1. Pathology in the cervical (CER; A), thoracic (THO; B), lumbar (LUM; C), and sacral (SAC; D) spinal cord of an 82-day-old G1H mouse. More NF spheroids are seen in sections of the cervical and thoracic spinal cord than in the lumbar and sacral spinal cord (arrowheads in A-C). The sections have been stained with anti-NFL polyclonal antiserum (A and D) and RM024 mAb (B and C) and counterstained with hematoxylin. Vacuoles are also present at each level of the spinal cord (see arrows in B and C). A-D are at the same magnification (see bar in D).
Proc. Natl. Acad. Sci. USA 93 (1996)
Neurobiology: Tu et al.
Interestingly, although vacuoles were detected in 82-day-old G1H mice (Fig. 1), no obvious vacuolar lesions were seen in the N1029 transgenic mice at 130 days (Fig. 3). Very mild vacuolar change was identified in the 199-day-old N1029 mice. Mild vacuolar change also was reported in the 250-day-old N1029 mice (17). However, these mice did develop NF-rich spheroids in their spinal cords at much later time points than the G1H mice (see Fig. 3). For example, NF-rich spheroids were first identified in the spinal cords of the N1029 transgenic mice at 132 days. The numbers of these NF-rich spheroids in 132-dayold N1029 mice were similar to those in 82-day-old G1H mice, and the spheroid numbers in 199-day-old N1029 mice were similar to the 130-day-old G1H mice (Table 1). However, the size, morphology, composition, and location of the spheroids in the N1029 mice were similar to those in the equivalent G1H mice. Intraneuronal Spheroids Contain All Three NF Subunits Including Phosphorylated Isoforms. The NF-rich spheroids contained all three NF subunits-i.e., the low (NFL), middle (NFM), and high (NFH)-molecular-weight NF proteins as revealed by immunohistochemistry using polyclonal and monoclonal (mAb) antibodies to each of these NF subunits (Fig. 2). Most NF-rich spheroids were homogeneously stained by antibodies to NF subunits, but occasionally a heterogeneous staining pattern was identified (Fig. 2 C and D). This result suggests that both assembled and unassembled or altered NF subunits are present in these spheroids. Furthermore, phosphorylation-dependent NFM and NFH antibodies stained the majority of the spheroids, which suggests that these spheroids contain phosphorylated forms of NFM and NFH (Fig. 2 H and
RESULTS NF-Rich Spheroids Occur in Both G1H and N1029 SOD1 from the G1H mice were examined, NF-rich spheroids similar to those seen in human ALS were identified in the 82-day-old (Fig. 1) and older G1H transgenic mice using antibodies to NF proteins. Further, the number of these NF inclusions increased with age (Table 1). This result suggests that the accumulation of these NF-rich spheroids is an age-dependent, progressive process. In the G1H mice, the initial accumulation of spheroids correlated with disease onset. More spheroids were seen in cervical and thoracic regions compared to lumbar and sacral spinal cord in the 82-day-old G1H mice, but older G1H mice contained similar numbers of these spheroids at all spinal cord levels. This result suggests that rostral spinal cord levels develop this pathology earlier or that they are more affected at this early disease stage. At all spinal cord levels, NF-rich spheroids were much more frequently found in the anterior horn and in the anterior and lateral columns of the white matter than in the posterior horn (Fig. 2). These findings are consistent with a disease process that selectively involves motor neurons. Degenerating motor neurons filled with perikaryal vacuoles were identified in the anterior and lateral horns of the spinal cord (Fig. 2A). However, NF-rich inclusions were most prominent in the processes of motor neurons, and they were rarely found in cell bodies (Fig. 2 C-L). Thickened dystrophic neurites filled with immunoreactive NFrich inclusions (many of which resembled spheroids) also were identified in the spinal cords of the G1H mice (Fig. 2B).
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3158
Neurobiology: Tu et al.
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J-L). Occasionally, highly phosphorylated NFM and NFH proteins accumulated in the perikarya of some motor neurons (data not shown).
spheroids were strongly stained by the antibodies to a-internexin and peripherin. Examination of a series of adjacent sections stained with antibodies to NF subunits and the other two neuronal IF proteins revealed that a-internexin and peripherin coexisted with NF proteins in some of these spheroids. For example, Figs. 3 F and G demonstrate the colocalization of NFL and a-internexin in the same spheroids. Double labeling also confirmed the colocalization of NF and the other two neuronal IF proteins in some spheroids, although most spheroids were only labeled by the antibodies to NF proteins (data not shown). Because antibodies to tubulin and actin failed to stain any of these spheroids (data not shown), it is likely
Other Neuronal IF Proteins but Not Tubulin or Actin Coexist in the NF-Rich Spheroids. Other neuronal IFs (i.e., a-internexin and peripherin) were normally expressed in selected populations of neurons and axons in the spinal cord. Because immunoreactive a-internexin and peripherin coexist in some NF-rich spheroids of patients with ALS (26, 27), we asked whether these neuronal IF proteins also contributed to the formation of the NF-rich spheroids in the transgenic mice. As shown in Fig. 3B, C, G, and H, some of the NF-rich
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