Relationship between neuropathology and disease progression in the SOD1G93A ALS mouse

Experimental Neurology 227 (2011) 287–295

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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r

Relationship between neuropathology and disease progression in the SOD1G93A ALS mouse Wendy W. Yang a, Richard L. Sidman b, Tatyana V. Taksir a, Christopher M. Treleaven a, Jonathan A. Fidler a, Seng H. Cheng a, James C. Dodge a, Lamya S. Shihabuddin a,⁎ a b

Genzyme Corporation, 49 New York Ave, Framingham, MA 01701-9322, USA Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA

a r t i c l e

i n f o

Article history: Received 12 July 2010 Revised 9 November 2010 Accepted 29 November 2010 Available online 9 December 2010 Keywords: Amyotrophic lateral sclerosis Astroglia Microglia SOD1G93A mouse Motor neuron Ventrolateral thalamus Disease progression

a b s t r a c t Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons. However, recent reports suggest an active role of non-neuronal cells in the pathogenesis of the disease. Here, we examined quantitatively the temporal development of neuropathologic features in the brain and spinal cord of a mouse model of ALS (SOD1G93A). Four phases of the disease were studied in both male and female SOD1G93A mice: presymptomatic (PRE-SYM), symptomatic (SYM), endstage (ES) and moribund (MB). Compared to their control littermates, SOD1G93A mice showed an increase in astrogliosis in the motor cortex, spinal cord and motor trigeminal nucleus in the SYM phase that worsened progressively in ES and MB animals. Associated with this increase in astrogliosis was a concomitant increase in motor neuron cell death in the spinal cord and motor trigeminal nucleus in both ES and MB mice, as well as in the ventrolateral thalamus in MB animals. In contrast, microglial activation was significantly increased in all the same regions but only when the mice were in the MB phase. These results suggest that astrogliosis preceded or occurred concurrently with neuronal degeneration whereas prominent microgliosis was evident later (MB stage), after significant motor neuron degeneration had occurred. Hence, our findings support a role for astrocytes in modulating the progression of non-cell autonomous degeneration of motor neurons, with microglia playing a role in clearing degenerating neurons. © 2010 Elsevier Inc. All rights reserved.

Introduction Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by muscle weakness and atrophy due to the loss of motor neurons in the motor cortex, brainstem and spinal cord. Sporadic ALS (SALS) is the most commonly diagnosed form of ALS as patients with familial ALS (FALS) only account for approximately 10% of all cases. The most frequent cause of FALS is currently attributed to mutations in the gene encoding cytosolic copper–zinc superoxide dismutase (SOD1) (Gurney et al., 1994; Rosen et al., 1993; Wong et al., 1995). Pathological analyses indicated that motor neuron cell death, involving apoptotic and probably additional mechanisms (Guegan and Przedborski, 2003), was preceded by aberrant neurofilament distribution in neuronal cell bodies and axons (Dal Canto and Gurney, 1994; Howland et al., 2002; Julien, 2001; Leigh and Swash, 1991; Nagai et al., 2001), and by early formation of insoluble SOD1containing protein complexes (Johnston et al., 2000). The subsequent

⁎ Corresponding author. Fax: +1 508 271 4776. E-mail addresses: [email protected] (W.W. Yang), [email protected] (R.L. Sidman), [email protected] (T.V. Taksir), [email protected] (S.H. Cheng), [email protected] (J.C. Dodge), [email protected] (L.S. Shihabuddin). 0014-4886/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2010.11.019

formation of ubiquitin-positive aggregates was also observed in ALS patients (Watanabe et al., 2001) and in mouse models of the disease (Bruijn et al., 1997; Bruijn et al., 1998; Wong et al., 1995). However, how the formation of such aggregates contributes to motor neuron apoptosis or autophagy is still unclear. As is the case with other diseases that exhibit neuronal degeneration, ALS is associated with astrogliosis, and microglial activation (Kato et al., 2000; Leigh and Swash, 1991). Although the analysis of diseased human samples has helped improve our understanding of the etiology and pathogenesis of ALS, because these were invariably from autopsy samples, they precluded a study of the pathological changes that may have accompanied or triggered disease onset and progression. As such, studies examining the temporal changes in CNS pathology of mouse models of ALS could provide complementary data that might aid the identification and development of novel therapeutic strategies. Several transgenic rodent models of ALS harboring mutant human SOD1 have been generated. These include the G93A mouse (Dal Canto and Gurney, 1997), G37R mouse (Wong et al., 1995), G85R mouse (Bruijn et al., 1997), H46R/H48Q mouse (Wang et al., 2002), and H46R and G93A rats (Aoki et al., 2005; Nagai et al., 2001). SOD1G93A mice are the most widely studied as they share many of the pathological features observed in SALS and FALS patients. A key finding from the studies of these genetic models was that mutations in SOD1

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result in a toxic gain rather than a loss of function, which is consistent with FALS being a dominant disease. Ubiquitous over-expression of mutant SOD1 in transgenic mice led to motor neuron death, whereas SOD1 knockout (SOD1-null) mice did not develop motor neuron pathology (Reaume et al., 1996). An emerging evidence suggests that mutant SOD1-mediated degeneration of motor neurons results from a combination of cell autonomous and non-cell autonomous processes, requiring the presence of mutant SOD1 in both neurons and glia. The evidence was initially accrued from the studies of human tissues and ALS mice showing that astroglial abnormalities and dysfunction often preceded clinical presentation of the disease (Bruijn et al., 1997; Howland et al., 2002; Maragakis and Rothstein, 2006). More recent studies with chimeric mice and cell cultures of normal and mutant motor neurons, as well as astrocytes and microglial cells showed that disease progression was most influenced by the presence of mutant astrocytes (Clement et al., 2003; Di Giorgio et al., 2007; Maragakis and Rothstein, 2006; Marchetto et al., 2008; Nagai et al., 2007). Taken together, the data suggest that the expression of mutant SOD1 in astrocytes and microglia contributes to the progression of ALS disease (Beers et al., 2006; Boillee et al., 2006b; Yamanaka et al., 2008b). Supposition was further buoyed by the demonstration that transgenic mice lacking mutant SOD1 selectively in astrocytes displayed a more delayed course of disease progression (Yamanaka et al., 2008b). Reducing the levels of mutant SOD1 within microglia also slowed disease progression in the SOD1G37R mice (Boillee et al., 2006b). However, a recent report showed that ablating proliferating microglia in the SOD1G93A mice did not arrest motor neuron degeneration, but decreased astrogliosis in the spinal cord (Gowing et al., 2008). Similarly, selective ablation of proliferating astrocytes did not affect disease outcome or motor neuron loss in SOD1G93A mice (Lepore et al., 2008a). Despite the accumulating evidence in favor of a contributory role of neighboring astrocytic and inflammatory non-neuronal cells in mutant SOD1mediated toxicity, the exact role of glia, and the exogenous factors that they release or that motor axons carry retrogradely to influence neurodegeneration remain to be further defined. To better understand the role of glial activation and how it correlates with motor neuron degeneration during disease progression, we characterized quantitatively the temporal progression of neuropathological changes at different stages of disease in the SOD1G93A mice. Astrocytic and microglial activation are well accepted features of ALS mice; however the progression of glial activation and other pathological features as a function of the disease has not been examined in detail in previous neuropathological studies (Boillee et al., 2006b; Hall et al., 1998). Previous studies had reported significant astroglial and microglial activation in the spinal cord of SOD1G93A and the loss of up to 50% of motor neurons at later stages of the disease (Chiu et al., 1995; Hall et al., 1998). More recent studies have also evaluated the role of astroglial (Keller et al., 2009), and microglial activation (Boillee et al., 2006b; Gowing et al., 2008) in the spinal cord as a function of disease progression. The present study extends these earlier findings by documenting the chronological progression of astroglial and microglial activation and correlating these changes to neuronal degeneration at different phases of the disease, in both the brain and spinal cord, of SOD1G93A mice. Materials and methods Animals Transgenic mice expressing the mutant human G93A SOD1 were used in our studies. These animals typically display disease onset at 90 days of age, and become moribund approximately 30 days later. Mice were divided into four groups, representing the different phases of disease: presymptomatic (age ~ 60 days), symptomatic (median age, males 85 ± 3 days, females 86 ± 3), end-stage (median age, males

108 ± 4, females 104 ± 3), and moribund (median age, males 121 ± 4, females 131 ± 4). There was no significant difference in the timing of the presentation of presymptomatic, symptomatic or end-stage phase of the disease between male and female mice. However, a difference in the age of male and female mice presenting the moribund phase of the disease was noted. Age-matched C57BL/6 non-transgenic littermates were used as wild-type controls. The following ALS scoring system was used to classify animals into the above phases: presymptomatic (PRESYM) = no visible motor abnormalities (i.e., normal hind limb splay); Symptomatic (SYM) = abnormal hind limb splay; End stage (ES) = onset of limb paralysis (typically hind limb); and Moribund (MB) = unable to right themselves within 30 s. Animals were maintained under a 12 h/12 h light/dark cycle and given water and food ad libitum. All procedures were performed using protocols that had been approved by the Institutional Animal Care and Use Committee. Tissue processing At sacrifice, all mice were deeply anesthetized with euthasol (150 mg/kg delivered intraperitoneally) and transcardially perfused with 0.1 M of phosphate buffered saline (PBS), followed by 4% buffered paraformaldehyde as described previously (Shihabuddin et al., 2004). Brains and spinal cords were promptly removed, post-fixed in the same fixative for 48 h at 4 °C, and then immersed in 30% sucrose until equilibration was reached. Brains and spinal cords were cryostatsectioned in the coronal plane at 20 μm and 10 μm, respectively. Immunohistochemistry Frozen brain and spinal cord sections were stained with the following antibodies: rabbit anti-GFAP (1:2500, DAKO, Glostrup, Germany), rabbit anti-neuronal nitric oxide synthase (1:1000, Millipore, Billerica, MA), rat anti-F4/80 (1:10, Genzyme, Cambridge, MA), or mouse anti-nitrotyrosine (1:500, Millipore, Billerica, MA). Secondary antibodies used were donkey anti-species specific antibodies that were conjugated with FITC or Cy3. Fluoro-Jade C (Histo-Chem Inc., Jefferson, AR) was used to detect degenerating neurons. Sections were visualized with a Nikon Eclipse E800 fluorescent microscope. Metamorph analysis A MetaMorph Image Analysis System (Universal Imaging Corporation, Downingtown, PA) was adapted to examine the staining intensity in specific areas of the brain and the entire gray matter of the spinal cord using 10x images of the described areas (Shihabuddin et al., 2004). The percentage increase in staining was determined by comparing the intensity of staining on diseased and wild type sections. A blinded examiner using a minimum of 10 sections per group performed the analysis. Cell count Cell counting was performed using the same MetaMorph Image Analysis System described above. Briefly, 20 μm spinal cord lumbar ventral horn area images were taken at the same exposure and set at the same threshold level. The threshold area of the entire image was first measured. The average individual cell threshold area was then determined, and the cell number was calculated by dividing the entire image threshold area by the average individual cell threshold area. Two sections per animal and 4 animals per group were examined in a blinded manner. Statistics The one-way ANOVA test was used to compare the differences between each diseased group and the wild-type group. For post-hoc

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analysis, Dunnett's multiple comparison test was used. Statistical significance was achieved when the p value was less than or equal to 0.05. All statistical analyses were performed with PRISM 4 (GraphPad Software, Inc., San Diego, CA). Results Time course of astrocytic activation Female and male SOD1G93A mice of different ages and representing the four different phases of disease (pre-symptomatic, symptomatic, end-stage and moribund) were killed and sections from their spinal cords and brains were processed for glial fibrillary acidic protein (GFAP) staining. Evidence of astrogliosis, an increase in GFAP staining indicative of astroglial hypertrophy and hyperplasia, was observed in all regions of the spinal cord (cervical, thoracic, lumbar and sacral) beginning in pre-symptomatic (PRE-SYM) mice and became progressively worse in the older animals (Fig. 1). As the intensity and extent of GFAP staining was noted to be equal throughout the cervical, thoracic, lumbar and sacral segments of the spinal cord, only those from the lumbar region are illustrated. Similarly, as the data from male and female SOD1G93A mice at each of the four phases of the disease were indistinguishable, the data from animals of both genders were combined. Metamorph analysis of the entire sections of the spinal cord showed that although GFAP staining was higher in the spinal cords of PRE-SYM mice, these did not reach statistical significance when compared to the signal from wild type control mice (Fig. 1B). However, those from symptomatic (SYM), end-stage

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(ES) and moribund (MB) animals were significantly higher than those from wild type mice (Table 1 and Fig. 1B). The increase in staining intensity from the ES and MB mice were approximately 8- (p b 0.01), and 10-fold (p b 0.01) higher, respectively. The observed increase in GFAP staining intensity was an aggregate of a higher signal in individual cells and an increase in the number of GFAP-positive cells (Fig. 1A, Table 2), however the relative contribution from each source was not determined. Of the brain sections analyzed, which included the motor trigeminal nucleus, motor cortex, ventrolateral thalamus, hippocampus and lateral septal nucleus, only those from the motor trigeminal nucleus of the brain stem and primary motor cortex (at bregma) showed an increase in GFAP staining intensity when compared to wild type controls (Table 1). Evidence of astrogliosis in the brain stem and motor cortex was apparent in SYM mice and was increased dramatically in ES and MB mice compared to wild type animals (Figs. 1A, B and Table 1). In the motor trigeminal nucleus, the staining intensity was increased 44- (p b 0.01), and 42-fold (p b 0.01) in ES and MB mice, respectively. In the primary motor cortex at bregma, GFAP staining intensity in the ES and MB mice were increased 8-, and 16fold (p b 0.01), respectively over control animals (Table 1). Time course of microglial activation Sections from the spinal cord and brain were also stained with an antibody against F4/80 to detect the presence of activated microglial cells. Evidence of microglial activation or microgliosis, enlarged microglial soma with the shortening and thickening of processes,

Fig. 1. GFAP Immunostaining and Quantification. Expression of astrocyte-specific marker, glial fibrillary acidic protein (GFAP) in the spinal cord and brain stem of SOD1G93A mice at different stages of the disease. A: Transverse sections of the lumbar spinal cord and lateral medulla in the brainstem showing GFAP immunostaining in SOD1G93A mice at different disease stages. GFAP positive cells were significantly increased at end stage and moribund phases. Scale bar: 100 μm. B: Quantitation of GFAP staining at different disease stages in SOD1G93A mice by MetaMorph analysis. Wild-type animals were used as controls, and integrated intensity of GFAP staining at different disease stages were compared with the controls. Values represent the mean ± SEM from 10 animals per stage. The numbers indicate fold-increases compared to wild-type mice (defined at 1.00 intensity). *p b 0.01. C: High magnification images showing the morphology of normal and activated astrocytes. Scale bar: 16 μm.

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Table 1 Quantitative evaluation of increases in various neuropathological markers in multiple regions of the CNS.

Lumbar

Motor trigeminal nucleus

Motor cortex (at bregma)

Ventrolateral thalamus

Hippocampus

Lateral septal nucleus

Disease stage

GFAP

F4/80

NOS

Nitrotyrosine

Fluoro-Jade C

PRE-SYM SYM ES MB PRE-SYM SYM ES MB PRE-SYM SYM ES MB PRE-SYM SYM ES MB PRE-SYM SYM ES MB PRE-SYM SYM ES MB

2.56 4.60⁎⁎ 8.06⁎⁎ 9.81⁎⁎ 1.28 7.21 43.66⁎⁎ 41.77⁎⁎

0.47 12.64 4.58 50.45⁎⁎ 0.14 1.56 5.98 42.06⁎⁎

4.06 5.04 19.16⁎⁎ 5.74 0.27 1.80 12.42 23.86⁎⁎

4.36 15.51 34.73 97.08⁎⁎ NC

0.72 2.17 23.87⁎⁎ 38.47⁎⁎ 1.04 71.19 150.04⁎ 147.20⁎

1.28 2.16 7.64 16.38⁎⁎ NC

NC

0.64 2.19 5.96 18.76⁎⁎ NC

NC

NC

NC

NC

1.27 1.34 3.25 48.87⁎ 0.66 1.67 15.27 44.44⁎⁎

1.02 2.14 30.17 169.01⁎⁎ NC

NC

0.20 0.46 1.35 14.07⁎⁎ NC

NC

NC

NC

NC

The numbers indicate fold-increases compared to wild-type mice (defined at 1.00 intensity). NC: No change between mutant and wild-type control mice. ⁎⁎ P b 0.01 versus wild-type control mice. ⁎ P b 0.05 versus wild-type control mice.

was noted in the gray matter of the spinal cords of mice at the SYM stage of the disease but the staining intensity (measured using Metamorph) did not reach statistical significance until the mice were at the MB phase when compared to the wild type animals (Figs. 2A and B). The spinal cord of mice in the MB phase showed a 30- to 50fold (p b 0.01) greater staining intensity compared to the wild-type controls (Table 1, Fig. 2B). The observed increase in staining intensity was an aggregate of a higher signal in individual cells and an increase in the number of F4/80 expressing cells (Fig. 2A, Table 2), however the relative contribution from each source was not determined. In the ventral horns of the spinal cord, there was a significant increase in the number of microglia at SYM, ES and MB when compared to wild type mice. It is noteworthy that at MB, the increase in cell number was almost triple those observed in SYM and ES animals. In the brain, F4/80 expressing activated microglial cells were detected in the motor trigeminal nucleus of the brainstem and in the ventrolateral thalamus initially in mice at the ES phase and became highly significant in the MB phase (Fig. 2B and Table 1). Moribund SOD1G93A mice showed a 42-fold (p b 0.01) higher level of staining intensity in the brain stem and a 14-fold (p b 0.01) higher signal in the ventrolateral thalamus compared to control mice. Hence, the advent of microglial activation would appear to occur later and only after astrogliosis became evident.

presence of degenerating neurons (Schmued et al., 2005). Our study is the first to report disease related changes in FJC staining that occur in ALS mice. Whether or not similar changes in FJC staining occur in ALS patient tissue remains to be determined. All levels of the spinal cord showed some measure of neurodegeneration at the SYM phase that became progressively and significantly more severe at the ES and MB phases (Figs. 3A, B). This is consistent with the results by Chiu et al. (1995) who showed progressive vacuolar changes in cholinergic motor neurons in the spinal cord of SOD1G93A mice, and a significant loss of these neurons by 90 days of age (onset of clinical symptoms). The FJC staining intensity at the ES phase (as illustrated in the lumbar region) was 24-fold (p b 0.01) and at the MB phase, 38-fold (p b 0.01) higher than in control mice (Table 1). Of the brain regions analyzed, only those of the motor trigeminal nucleus and ventral lateral thalamus stained positively with FJC. As noted above for the spinal cord, evidence of neurodegeneration was apparent in the brain stem in the SYM phase. However, the extent of degeneration was significantly greater in the ES (150-fold, p b 0.05) and MB (148-fold, p b 0.05) phases compared to control mice (Fig. 3B and Table 1). The staining in the ventral lateral thalamus was also significantly elevated (169-fold, p b 0.01) but only in MB mice (Table 1). Time course of expression of nitric oxide synthase and nitrotyrosine

Time course of neurodegeneration Sections of spinal cords and brains from SOD1G93A mice at the four different phases were stained with fluoro-Jade C (FJC) to detect the Table 2 Cell counts of astrocytes and microglia in the ventral horn of the lumbar spinal cord at different stages of the disease.

GFAP F4/80

WT

PRE-SYM

SYM

ES

MB

2 ± 0.2 3 ± 2.3

4 ± 0.4 2 ± 0.4

39 ± 2.8⁎⁎ 37 ± 6.8⁎⁎

99 ± 5.1⁎⁎ 44 ± 2.6⁎⁎

118 ± 7.8⁎⁎ 128 ± 4.8⁎⁎

⁎⁎ P b 0.01 versus wild-type control mice.

Nitric oxide (NO) reportedly contributes to the pathogenesis of ALS (Catania et al., 2001) through its reaction with superoxide anions to generate peroxynitrite, a potent oxidant and inhibitor of the mitochondrial electron transport chain (Urushitani and Shimohama, 2001). Peroxynitrite mediates tyrosine nitration to generate nitrotyrosine, which has been shown to be elevated in the CNS of ALS patients and mouse models of the disease (Beal et al., 1997; Cha et al., 2000; Ferrante et al., 1997). The staining of the tissue sections from SOD1G93A mice for nitric oxide synthase (NOS) showed that the signal was primarily colocalized with GFAP-positive cells (data not shown). Increased intensity of NOS staining was observed throughout the spinal cord

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Fig. 2. F4/80 Immunostaining and Quantification. Expression of microglia-specific marker, F4/80, in the spinal cord and brainstem of SOD1G93A mice at different stages of the disease. A: Transverse sections of the lumbar spinal cord and lateral medulla of the brainstem showing F4/80 immunostaining in SOD1G93A mice at different disease stages. F4/80 positive cells were significantly increased at moribund phase. Scale bar: 100 μm. B: Quantitation of F4/80 staining at different disease stages in SOD1G93A mice by MetaMorph analysis. Wildtype animals were used as controls, and integrated intensity of F4/80 staining at different disease stages were compared with the controls. Values represent the mean ± SEM from 10 animals per stage. The numbers indicate fold-increases compared to wild-type mice (defined at 1.00 intensity). *p b 0.01. C: High magnification images showing the morphology of normal and activated microglia. Scale bar: 16 μm.

starting at the PRE-SYM disease stage (Fig. 4). Metamorph analysis showed that the NOS staining were significantly higher in ES mice (p b 0.01) compared to wild type mice but was reduced in MB animals (Fig. 4B and Table 1). In the brain, significantly increased NOS staining was evident in the motor trigeminal nucleus of the brainstem (24fold, p b 0.01) and in the primary motor cortex at bregma (19-fold, p b 0.01) in MB animals (Table 1). As in the spinal cord, there were greater NOS staining in PRE-SYM mice (2-fold) that clearly increased in intensity at the later stages of the disease. Sections were also probed for immunoreactivity to nitrotyrosine as a marker of peroxynitrite. In the lumbar spinal cord, nitrotyrosine levels were significantly elevated in ES (35-fold) and MB (97-fold) mice when compared to control animals (Fig. 4A and Table 1). However, in the brain, the staining pattern was different from that for NOS. Staining for nitrotyrosine was increased in the hippocampus and lateral septal nucleus but not in the brain stem or motor cortex. The most profound changes were noted in MB mice with the hippocampus exhibiting a 49-fold (p b 0.05) and the lateral septal nucleus exhibiting a 44-fold (p b 0.01) higher level of staining than in control animals (Table 1). Discussion Over the last decade, the evidence from the studies of transgenic mouse models has strongly suggested a role of non-neuronal cells in the pathogenesis of ALS (Boillee et al., 2006a; Yamanaka et al., 2008b). However, the influence of astroglia and microglia in ALS as well as other disorders of the CNS remained ambiguous as these cells purportedly could display both neuroprotective and neurotoxic

activities (Hall et al., 1998; McGeer and McGeer, 1995; Minghetti and Levi, 1998; Sargsyan et al., 2005). In the present study, we documented the time course of neuropathological changes in the brain and spinal cord of SOD1G93A ALS mice and examined their correlation with disease progression. Early studies of transgenic mice that selectively expressed mutant SOD1 in neurons showed a lack of motor neuron degeneration (Lino et al., 2002; Pramatarova et al., 2001). A more recent report showed that when the expression of mutant SOD1 was restricted to motor neurons, or to motor neurons and oligodendrocytes, it was sufficient to cause motor neuron disease; however, the onset and severity of the disease was significantly delayed and attenuated in the presence of wild type neighboring cells (Jaarsma et al., 2008; Yamanaka et al., 2008a). This is in accord with other studies showing that downregulation of mutant SOD1 specifically in neurons delayed the onset of symptoms and prolonged survival (Boillee et al., 2006b). Furthermore, chimeric mouse models of ALS harboring increasing amounts of wild-type non-neuronal cells displayed progressively less severe disease phenotypes (Clement et al., 2003). These data support the notion that the toxic activity of mutant SOD1 in neurons contributes to the pathogenesis of ALS and that neighboring glial cells can be significant contributors to the neuronal degenerative disease. However, confounding this supposition are the reports that astrocytes can also exhibit a neuroprotective effect in neurodegenerative diseases (Hall et al., 1998; McGeer and McGeer, 1995) by promoting motor neuron survival and repair (Ilieva et al., 2009; Staats and Van Den Bosch, 2009). Our study of the SOD1G93A mouse showed that astrogliosis was evident in both the brain and spinal cord as early as the symptomatic

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Fig. 3. Fluoro-Jade C Staining and Quantification. Expression of cell death marker, Fluoro-Jade C (FJC), in the spinal cord and brainstem of SOD1G93A mice at different stages of the disease. A: Transverse sections of the lumbar spinal cord and lateral medulla of the brainstem showing FJC staining in SOD1G93A mice at different disease stages. FJC positive cells were significantly increased at the end stage and the moribund phases. Scale bar: 100 μm. B: Quantitation of FJC staining at different disease stages in SOD1G93A mice by MetaMorph analysis. Wild-type animals were used as controls and integrated intensity of FJC staining at different disease stages were compared with the controls. Values represent the mean ± SEM from 10 animals per stage. The numbers indicate fold-increases compared to wild-type mice (defined at 1.00 intensity). *p b 0.01. C: High magnification image showing positive FJC-stained cells. Scale bar: 16 μm.

and presymtomatic phase, respectively. By end-stage, when motor neurons were being lost (Chiu et al., 1995; Dal Canto and Gurney, 1994; Gurney et al., 1996; Gurney et al., 1994), the number of activated astrocytes were significantly increased in the spinal cord, the motor trigeminal nucleus of the brainstem, and the primary motor cortex. These results suggest that astrocytes might have been activated during the early phases of motor neuron degeneration and disease progression to perhaps nourish and repair the functionally declining neuronal cells. The neuroprotective role of astrocytes is supported by a recent study showing that astrocyte replacement extended survival and disease duration in SOD1G93A rat model of ALS attenuated (Lepore et al., 2008b). At later stages of the disease, the impact of this cell type might have become more explicitly negative. That astrocytes could adversely exacerbate the disease is supported by the study of Yamanaka et al. (2008b) showing that a reduction of mutant SOD1 selectively in astrocytes slowed disease progression. Treatment of ALS mice with certain neurotrophic factors that prolonged disease progression was also associated with a decline in GFAP expression (Dodge et al., 2008). Microglia play a critical role as resident immune cells and pathological sensors within the CNS. Resting microglial cells have a small cell body and fine ramified processes and are highly sensitive to changes in the tissue microenvironment. Activated microglia display thickening and shortening of cellular processes as well as increased expression of activation markers (Sargsyan et al., 2005). Microglial activation could be caused by numerous neuronal stresses including loss of motor neurons or astrocytic activation (Gowing et al., 2008; Magnus et al., 2008). Activated microglia could in turn produce various cytotoxic mediators including oxygen radicals, nitric oxide,

peroxynitrite, and glutamate, and it is reasonable that these cytotoxic products might cause neuronal injury (Banati et al., 1994; Gehrmann et al., 1995; Holevinsky and Nelson, 1995; Hu et al., 1996; Hu et al., 1995; Yoshida et al., 1995). Nitric oxide has also been implicated as a mediator of neuron cell death, through its promotion of peroxynitrite formation, in several neurodegenerative disorders including ALS (Beckman et al., 1993; Cha et al., 1998). Reports of increased nitrotyrosine-related immunoreactivity (a marker of peoxynitrite) in upper and lower motor neurons from ALS patients (Abe et al., 1995; Beal et al., 1997) and in SOD1G93A mice (Hall et al., 1998) have provided evidence for the role of nitric oxide and peroxynitrite in motor neuron disease. In ALS, strong activation of microglia has been reported to occur in regions of motor neuron loss (Henkel et al., 2004). Similar to the earlier studies, we also showed that microglial activation was initiated at or soon after the disease onset in mutant SOD1G93A mice (Alexianu et al., 2001; Hall et al., 1998; Kriz et al., 2002). This was evidenced by the increase in the numbers of activated microglia expressing F4/80, and displaying thickened and shortened processes, as the disease progressed. Significant microglial activation was seen only in those regions where neurodegeneration was well advanced, as indicated by Flourojade C staining. The significant increase in Fluorojade C staining preceded the increase in microglial activation in the spinal cord, motor trigeminal nucleus and ventrolateral thalamus, suggesting that significant microglial activation only occurred after the onset of cell death and astroctytic activation. In the ventral horns of the lumbar spinal cord, the increase in the number of microglia was seen starting at the symptomatic phase, and coincided with the initial appearance of neurodegeneration, as evidenced by the increase in Fluorojade C staining. By the MB stage, the number of

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Fig. 4. NOS Immunostaining and Quantification. Expression of nitric oxide synthase (NOS) activity and peroxynitrate (Nitrotyrosine) formation in the spinal cord of SOD1G93A mice at different stages of the disease. A: Transverse sections of the lumbar spinal cord showing NOS and Nitrotyrosine immunostaining in SOD1G93A mice at different disease stages. NOS positive cells were significantly increased at the end stage, and Nitrotyrosine positive cells were significantly increased at the moribund phase. Scale bar: 100 μm. B: Quantitation of NOS and Nitrotyrosine staining at different disease stages in SOD1G93A mice by MetaMorph analysis. Wild-type animals were used as controls and integrated intensity of NOS and Nitrotyrosine staining at different disease stages were compared with the controls. Values represent the mean ± SEM from 10 animals per stage. The numbers indicate fold-increases compared to wild-type mice (defined at 1.00 intensity). *p b 0.01. C: High magnification images of positive NOS- and Nitrotyrosine-stained cells. Scale bar: 16 μm.

activated microglia was significantly increased as compared to the SYM and ES phases and correlated with the ongoing neurodegeneration. It is worth noting that neuronal loss and microglial activation in the ventrolateral thalamus was not associated with astrogliosis. Whether this regional neuronal loss was a consequence of localized degeneration or loss of innervations from other regions of the CNS remains unclear. Our data support the recent findings that proliferating microglia might not be the key contributors to disease progression and motor neuron degeneration in SOD1 mice (Gowing et al., 2008). In agreement with the finding, a phase III randomized clinical trial testing the therapeutic potential of minocyline, a drug shown to inhibit microglia activation, accelerated rather than slowed disease deterioration in ALS patients (Gordon et al., 2007). It is possible that microglia might be acting partly as pathological sensors and scavengers to remove apoptotic cells at the later stages of the disease. However, activation of microglia might also provide a feedback loop to enhance activation of astrocytes. Supporting this view, a 50% decrease in activated microglia led to a reduction in activated astrocytes but without affecting motor neuron degeneration in transgenic SOD1 mice (Gowing et al., 2008). In contrast, Boillee et al. (2006b) showed that lowering mutant SOD1 expression within microglia significantly slowed disease progression without affecting disease onset. However, this study did not address the role of microglia per se in the disease process, but might reflect the presence of a toxic action of mutant SOD1 in microglia. This hypothesis was supported by studies showing a neuroprotective role of wild-type microglia in familial ALS (Beers et al., 2006), but that the expression of mutant SOD1 in microglia reduced their neurotrophic potential

and increased their neurotoxic potential as compared to wild-type microglia (Xiao et al., 2007). It is worth noting that in the present study we evaluated glial activation as a function of the onset of the four phases of the disease. Our data showed that there were astroglial and microglial activation at specific phases of the disease and that these changes were indistinguishable in male and female mice. The only observed gender difference was in the age of onset of MB, where females showed a trend towards longer survival than males after the onset of ES. Although previous studies had reported a sex dimorphic response to neuroprotective intervention in the SOD1G93A mice, the basis for the observed male selective benefit (Lepore et al., 2007) and in other instances, a female selective benefit (Teng et al., 2006) remains unclear. It appears to be dependent on the agents used in the studies and other factors such as the timing of the intervention and differences in the levels of sex hormones at the time of the study. The present study also showed that in the spinal cord NOS expression was elevated in PRE-SYM animals and significantly increased in animals at ES, but returned to SYM levels at the subsequent moribund phase. Nitric oxide can react with superoxide to form peroxynitrite resulting in the nitration of glutamate transporter proteins and mitochondrial respiratory chain, which inhibit their activity (Beal et al., 1997). In our study, peroxynitrite, visualized by nitrotyrosine staining, was also upregulated during the early phases of the disease; however, it continued to increase progressively with disease progression within the spinal cord. Therefore, a drop off in NOS expression in MB mice may be due to a negative feedback loop triggered by elevated peroxynitrite generation. Nevertheless, our findings, support a potential role of NOS and peroxynitrite in

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cytotoxicity and disease progression. Interestingly, NOS upregulation was only seen in regions that showed increased astrogliosis and in the motor cortex this was not associated with significant microglial activation or cell loss. In summary, astrocytic activation was seen early in the disease process and preceded microglial activation. The latter was only observed after onset of significant cell loss in the spinal cord and motor trigeminal nucleus of the SOD1G93A mice. However, the presence or absence of astrocytic activation in the motor cortex and ventrolateral thalamus, respectively, was not indicative of cell loss at the later stages of the disease. This data support the potential role of activated astrocytes in disease progression and the role of microglia as scavengers to remove cell debris. References Abe, K., Pan, L.H., Watanabe, M., Kato, T., Itoyama, Y., 1995. Induction of nitrotyrosinelike immunoreactivity in the lower motor neuron of amyotrophic lateral sclerosis. 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