Acta Neuropathologica

, Volume 120, Issue 1, pp 75–84

TDP-43 pathology in sporadic ALS occurs in motor neurons lacking the RNA editing enzyme ADAR2

Authors

  • Hitoshi Aizawa
    • Division of Neurology, Department of Internal MedicineAsahikawa Medical College
  • Jun Sawada
    • Division of Neurology, Department of Internal MedicineAsahikawa Medical College
  • Takuto Hideyama
    • CREST, Japan Science and Technology Agency, Department of Neurology, Graduate School of MedicineThe University of Tokyo
  • Takenari Yamashita
    • CREST, Japan Science and Technology Agency, Department of Neurology, Graduate School of MedicineThe University of Tokyo
  • Takayuki Katayama
    • Division of Neurology, Department of Internal MedicineAsahikawa Medical College
  • Naoyuki Hasebe
    • Division of Neurology, Department of Internal MedicineAsahikawa Medical College
  • Takashi Kimura
    • Department of NeurologyDouhoku National Hospital
  • Osamu Yahara
    • Department of NeurologyDouhoku National Hospital
    • CREST, Japan Science and Technology Agency, Department of Neurology, Graduate School of MedicineThe University of Tokyo
Original Paper

DOI: 10.1007/s00401-010-0678-x

Cite this article as:
Aizawa, H., Sawada, J., Hideyama, T. et al. Acta Neuropathol (2010) 120: 75. doi:10.1007/s00401-010-0678-x

Abstract

Both the appearance of cytoplasmic inclusions containing phosphorylated TAR DNA-binding protein (TDP-43) and inefficient RNA editing at the GluR2 Q/R site are molecular abnormalities observed specifically in motor neurons of patients with sporadic amyotrophic lateral sclerosis (ALS). The purpose of this study is to determine whether a link exists between these two specific molecular changes in ALS spinal motor neurons. We immunohistochemically examined the expression of adenosine deaminase acting on RNA 2 (ADAR2), the enzyme that specifically catalyzes GluR2 Q/R site-editing, and the expression of phosphorylated and non-phosphorylated TDP-43 in the spinal motor neurons of patients with sporadic ALS. We found that all motor neurons were ADAR2-positive in the control cases, whereas more than half of them were ADAR2-negative in the ALS cases. All ADAR2-negative neurons had cytoplasmic inclusions that were immunoreactive to phosphorylated TDP-43, but lacked non-phosphorylated TDP-43 in the nucleus. Our results suggest a molecular link between reduced ADAR2 activity and TDP-43 pathology.

Keywords

Amyotrophic lateral sclerosisAdenosine deaminase acting on RNA 2TDP-43RNA editingMotor neuron

Introduction

Amyotrophic lateral sclerosis (ALS) is a devastating disease characterized by a progressive deterioration of motor function resulting from the degeneration of motor neurons. More than 90% of ALS cases are sporadic and approximately 5–10% are familial. Although at least six causal genes have been identified so far in individuals affected with familial ALS, SOD1 [30], ALS2 (alsin) [10, 39], senataxin (ALS4) [7], vesicle-trafficking protein/synaptobrevin-associated membrane protein [27], TAR DNA-binding protein (TDP-43) [9, 15, 32, 37, 40] and FUS/TLS [22, 38], the pathogenesis of sporadic ALS remains largely unexplored.

One hypothesis for selective neuronal death in sporadic ALS is excitotoxicity mediated by abnormally Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, which are a subtype of the ionotropic glutamate receptor [6, 20, 21]. The recent finding of reduced RNA editing of the AMPA receptor subunit GluR2 at the Q/R site provides a plausible pathogenic mechanism underlying motor neuron death in sporadic ALS [16, 21, 34]. Reduced GluR2 Q/R site-editing, catalyzed by an enzyme called adenosine deaminase acting on RNA 2 (ADAR2), appears to be specific to sporadic ALS among several neurodegenerative diseases [1, 19, 20, 29, 33].

TDP-43 was identified as a component of ubiquitin-positive, but tau-negative cytoplasmic inclusions in cortical neurons in patients with frontotemporal lobar degeneration (FTD) and spinal motor neurons in patients with sporadic ALS [3]. The TDP-43 found in these inclusions was demonstrated to be abnormally phosphorylated [12, 26].

Because both reduced GluR2 Q/R site-editing and formation of TDP-43-containing inclusions occur specifically in sporadic ALS motor neurons, we used immunohistochemistry to examine the expression of TDP-43 and ADAR2 in ALS motor neurons and elucidate a link between these two molecules.

Materials and methods

Subjects

This study was conducted using lumbar spinal cords from seven cases of sporadic ALS and six disease-free control cases. Consent for autopsy and approval for the use of human tissue specimens for research purposes was approved by appropriate institutional human ethics committees. Clinical information is given in Table 1.
Table 1

Profiles of ALS cases and disease controls

Case

Age

Sex

PMI (h)

Brain weight (g)

Diagnosis

Duration of ALS

Onset of ALS

Number of MN/AH mean ± SD

ADAR2 (+) pTDP-43 (−) MN/AH mean ± SD (%)

ADAR2 (−) pTDP-43 (+) MN/AH mean ± SD (%)

ADAR2 (+) pTDP-43 (+) MN/AH n (%)

Number of AH examined

1

67

M

3

1,390

ALS

1 year 1 month

UE

6.0 ± 2.6

1.3 ± 1.5 (20.8)

4.8 ± 2.4 (79.2)

0

4

2

59

F

5

1,290

ALS

1 year 9 months

UE

2.3 ± 1.2

0 ± 0 (0.0)

2.3 ± 0.5 (100)

0

4

3

69

M

3

1,430

ALS

2 years

UE

3.3 ± 1.5

2.3 ± 1.9 (69.6)

1.0 ± 0.8 (30.4)

0

7

4

75

F

2

1,080

ALS

2 years 6 months

LE

8.7 ± 3.1

5.7 ± 2.8 (65.4)

3.0 ± 3.0 (34.6)

1 (0.2)

6

5

72

M

1.5

1,290

ALS

3 years 1 month

Bulb

3.5 ± 1.6

0.5 ± 0.6 (14.3)

3.0 ± 1.2 (85.7)

0

4

6

62

M

3

1,390

ALS

3 years 7 months

UE

4.9 ± 1.7

1.3 ± 0.8 (26.5)

3.6 ± 1.7 (73.5)

0

7

7

69

M

1.5

1,460

ALS

12 years

UMN

2.2 ± 0.7

0.8 ± 0.4 (38.5)

1.3 ± 0.3 (61.5)

0

6

8

64

M

2

1,200

Cerebellar tumor

  

15.8 ± 8.6

15.8 ± 8.6 (100)

0

0

4

9

58

M

7

1,420

Myotonic dystrophy

  

13.3 ± 7.2

13.3 ± 7.2 (100)

0

0

4

10

70

F

2

1,040

Limb-girdle muscular dystrophy

  

10.2 ± 5.4

10.2 ± 5.4 (100)

0

0

6

11

73

M

3

1,170

Theophylline intoxication

  

8.2 ± 4.9

8.2 ± 4.9 (100)

0

0

6

12

77

M

1.5

1,200

Limb-girdle muscular dystrophy

  

10.9 ± 6.1

10.9 ± 6.1 (100)

0

0

8

13

78

M

1

1,310

Meningitis

  

8.4 ± 4.6

8.4 ± 4.6 (100)

0

0

8

PMI postmortem interval, AH anterior horn, MN motor neuron, UE upper extremities, LE lower extremities, Bulb bulbar symptoms, UMN upper motor neuron sign

Western blot analysis using the anti-ADAR2 antibody (RED1)

To examine the specificity of the polyclonal anti-ADAR2 antibody (RED 1) (Exalpha Biologicals, Watertown, MA) in the human brain, Western blot analysis was performed as reported previously [17]. From 100 mg of human frontal cortex, nuclear and cytoplasmic fractions were separated with the PARIS Protein and RNA Isolation System (TAKARA, Tokyo) according to the manufacturer’s instructions. Nuclear and cytoplasmic proteins as well as those containing recombinant ADAR2a (rADAR2a) and recombinant ADAR2b (rADAR2b) proteins synthesized by in vitro translation were suspended in 500 μl of cold Cell Fraction Buffer provided with the PARIS Protein and RNA Isolation System (TAKARA). Samples were then boiled with 500 μl of 2 × SDS gel-loading buffer and subjected to SDS-PAGE. After electrophoresis, proteins were transferred to an Immobilon-P transfer membrane (Millipore, Bedford, MA). Blots were blocked in a buffer containing Tween/PBS and 1% bovine serum albumin (BSA). Then immunoblotting for histone protein (MAB052; CHEMICON, Temecula, CA, 1:2,000), glyceraldehyde-3 phosphate dehydrogenase (GAPDH) (MAB374; CHEMICON, 1:2,000) or ADAR2 (RED1; Exalpha Biologicals, Watertown, MA, 1:4,000) was conducted overnight at 4°C. For secondary antibodies, peroxidase-conjugated AffiniPure goat anti-mouse IgG (H + L) (Jackson ImmunoResearch, West Grove, PA; 1:5,000) or peroxidase-conjugated AffiniPure rabbit anti-sheep IgG (H + L) (Jackson ImmunoResearch; 1:5,000) was used. Visualization was carried out using ECL plus Western blotting detection reagents (GE Healthcare Bioscience, Piscataway, NJ). Specific bands were detected with an LAS 3000 system (Fujifilm, Tokyo).

Immunohistochemical analysis

The human spinal cords were fixed in 10% neutral buffered formalin for about 7 days and then embedded in paraffin. Serial 7 μm sections were cut for immunohistochemical analysis. The immunoreactive features of the anti-ADAR2 antibody on frozen sections were also evaluated. To examine the localization of ADAR2 and TDP-43 in a single neuron, a pair of adjacent sections was used for immunohistochemistry. The sections were mounted on slides and then deparaffinized in xylene, hydrated with an ethanol series and heated at 120°C for 2 min for antigen retrieval. The sections were then washed with phosphate-buffered saline (PBS) and incubated with the primary antibody overnight at 4°C. Polyclonal anti-ADAR2 (RED1, 1:100), monoclonal anti-phosphorylated TDP-43 (pTDP-43) (pS409/410) (Cosmo Bio Co., Ltd., Tokyo, Japan; 1:3,000) and rabbit polyclonal phosphorylation-independent anti-TARDBP (piTDP-43) (Protein-Tech Group, Inc.; 1:3,000) were used. Bound antibodies were detected using an avidin–biotin–peroxidase complex kit (Vector Laboratories, Burlingame, CA, USA). Diaminobenzidine tetrahydrochloride was used as the chromogen, and the sections were lightly counterstained with hematoxylin. The RED1 antibody was incubated with 5 μg/μl of recombinant ADAR2 (Abnova Corp., Taiwan) at 4°C overnight. These samples were then subjected to immunohistochemistry for the preabsorption test.

To test the effects of fixation, the paraffin-embedding procedure and the postmortem delay on ADAR2 immunoreactivity, rat spinal cord samples were processed either immediately after removal (PMI-0) or after the spinal cords were held at room temperature for 6 h (PMI-6) or 24 h (PMI-24). Some samples were quickly frozen on dry ice, and the others were fixed in 10% buffered formalin after each treatment. The immunohistochemical process was the same as that used for the paraffin-embedded human sections, except that frozen sections were incubated with the primary antibody for 1 h at room temperature after washing with PBS.

Frozen sections of lumbar spinal cords from SOD1G93A transgenic mice at 24 and 35 weeks of age were also used for immunohistochemistry with anti-ADAR2 and anti-pTDP-43 antibodies.

Double immunofluorescence study using anti-ADAR2 antibody and anti-phosphorylation-independent TDP-43 antibody

Formalin-fixed paraffin-embedded spinal cord sections from an ALS patient (case 6 in Table 1) were double-immunostained with RED1 antibody (×100) and anti-TDP-43 monoclonal antibody (Abnova, ×1,000). Labeled goat anti-rabbit IgG antibody (Molecular Probes, Alexa 488) and labeled goat anti-mouse IgG (Molecular Probes, Alexa 594) were used as secondary antibodies (×1,000).

Quantification of motor neurons in ALS and control spinal cords

Serial sections of both ALS and control cases were immunostained with piTDP-43, ADAR2, or pTDP-43. Large ADAR2-positive and -negative neurons with nucleoli in the anterior horns on each section were counted separately. In addition, we examined whether each of the motor neurons was immunostained with pTDP-43 or piTDP-43 in the respective adjacent section.

Statistics

The Mann–Whitney U test was used to compare the number of anterior horn cells (AHC) in ALS samples compared to controls.

Results

Nuclear localization of ADAR2

The Western blot analysis of the human cortex showed that the anti-ADAR2 antibody (RED1) recognized two isoforms of active ADAR2 protein, ADAR2a and ADAR2b, in the nuclear fraction, but not in the cytoplasmic fraction. It is reasonable for ADAR2 to be localized in the nuclear fraction because ADAR2 primarily acts on RNA. The validity of this fractionation was verified by the presence of histone in the nuclear fraction and of GAPDH in the cytoplasmic fraction (Fig. 1a). ADAR2 immunoreactivity is observed in the nuclei of motor neurons from frozen sections of rat (Fig. 1b) and human spinal cords (Fig. 1c).
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Fig. 1

Nuclear localization of the ADAR2 protein in the human brain. Western blot analysis of the human cortex demonstrates that ADAR2 protein is localized in the nuclear fraction, but not in the cytoplasmic fraction. The validity of this fractionation was verified by the presence of histone in the nuclear fraction and of GAPDH in the cytoplasmic fraction. ADAR2 immunoreactivity is demonstrated in the nuclei of large neurons in the anterior horn of the rat spinal cord (b) and the human spinal cord (c). Bar indicates 20 μm. N nuclear fraction, C cytoplasmic fraction, H brain homogenate, rADAR2a recombinant human ADAR2a, rADAR2b recombinant human ADAR2b

ADAR2 expression in motor neurons of normal rat and SOD1 transgenic mouse

Intense ADAR2 immunoreactivity was observed in the nucleolus of the nuclei from all motor neurons examined in the frozen rat sections (Fig. 2a, c), whereas both the nucleus and cytoplasm were immunoreactive for ADAR2 in the paraffin-embedded sections of both PMI-0 and PMI-6 tissues (Fig. 2b, d, e, g, h). The intensity of ADAR2 immunoreactivity varied markedly among the nuclei (even on the same section) and was uniformly low in the cytoplasm of the motor neurons in the paraffin-embedded sections. ADAR2 immunoreactivity in the nuclei of motor neurons on the frozen and paraffin-embedded sections of PMI-24 tissue was less intense than in PMI-0 and PMI-6 tissues (Fig. 2). ADAR2 immunoreactivity was observed in the nucleus of all the motor neurons examined in the spinal cords of SOD1G93A transgenic mice (Fig. 3b).
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Fig. 2

ADAR2 immunohistochemistry of motor neurons in the rat lumbar spinal cord. ADAR2 is expressed in the nuclei of neurons in frozen sections created at 0 h postmortem (a, c). On the contrary, ADAR2 is always positive in the cytoplasm of neurons in formalin-fixed, paraffin-embedded sections from different rats, with a variable intensity of nuclear immunoreactivity among neurons (b, d, e). Frozen (f) and paraffin-embedded sections (g, h) with a 6-h postmortem interval display immunoreactivity similar to those with a 0-h postmortem interval. Nuclear immunoreactivity was less intense on both frozen sections (i) and paraffin-embedded sections (j, k) created at 24 h postmortem compared with those prepared at 0 h. PM postmortem interval. Bar indicates 20 μm

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Fig. 3

Expression of ADAR2 and phosphorylation-dependent TDP-43 in spinal motor neurons of SOD1 transgenic mice. A low-magnification view of the lumbar spinal anterior horn in a SOD1G93A transgenic mouse, at 34 weeks of age showing that all the motor neurons are ADAR2 positive (a). A high-magnification view shows predominant nuclear immunoreactivity (b). There is no phosphorylation-dependent TDP-43 immunoreactive inclusion in the motor neurons (c, arrow). Bar indicates 20 μm

ADAR2, phosphorylation-dependent TDP-43 and phosphorylation-independent TDP-43 expression in human control spinal motor neurons

In the spinal cords of human control cases, all motor neurons examined (n = 380 from 6 cases) showed ADAR2 immunoreactivity, typically in the cytoplasm with slight or no apparent immunoreactivity observed in the nuclei (Table 1; Figs. 4a, b, 5a). Similar ADAR2 immunoreactivity was observed in all the neurons in the pontine nuclei, including atrophic neurons in patients with multiple system atrophy and spinocerebellar atrophy type 1 (Supplementary Figure 1). Phosphorylation-independent TDP-43 (piTDP-43) stained the nuclei of the same motor neurons (Figs. 4a′, a″, b′, 5b, c), while phosphorylation-dependent TDP-43 (pTDP-43) did not stain either the nucleus or cytoplasm on the adjacent section (Fig. 4b″). Two different anti-ADAR2 antibodies exhibited immunoreactivity in the cytoplasm of motor neurons (Fig. 4a, b, g, Supplementary Figure 2), and preabsorbed anti-ADAR2 antibody did not show any immunoreactivity (Fig. 4h).
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Fig. 4

Expression of ADAR2, phosphorylation-dependent TDP-43 and phosphorylation-independent TDP-43 in spinal motor neurons from adjacent sections. A low-magnification view of the lumbar spinal anterior horn in a control subject (case 8) shows that all the motor neurons are immunoreactive for ADAR2 (a). These neurons show phosphorylation-independent TDP-43 (piTDP-43) immunoreactivity in the nucleus in an adjacent section (a′, a″: a high-magnification view of the open square in a′). A lumbar spinal motor neuron from a control subject (case 12) shows diffuse ADAR2 immunoreactivity in the cytoplasm (b). The adjacent section shows piTDP-43 immunoreactivity in the nucleus (b′), but does not show phosphorylation-dependent TDP-43 (pTDP-43) immunoreactivity (b″). All ADAR2-negative neurons (closed arrows in c; case 6) display pTDP-43-positive inclusions in the cytoplasm (c′), whereas an ADAR2-positive neuron from a patient with ALS (open arrow in c) has no pTDP-43 immunoreactivity (c′). An ADAR2-negative neuron (d; case 6) has multiple pTDP-43-positive inclusions in the cytoplasm (d′). ADAR2-positive neurons (e case 6, f case 3) show phosphorylation-independent TDP-43 immunoreactivity in the nucleus (e′, f′). ADAR2 immunoreactivity in the cytoplasm (g) disappeared when recombinant ADAR2-preabsorbed anti-ADAR2 antibody was used as the primary antibody (h) (case 12). Bar indicates 20 μm

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Fig. 5

Double-labeled immunofluorescence study using anti-ADAR2 and anti-piTDP-43 antibodies. ac Control motor neuron was immunopositive for ADAR2 in the cytoplasm and nucleus and piTDP-43 in the nucleus. df An ALS motor neuron immunoreactive to ADARA2 in the cytoplasm showed immunoreactivity to piTDP-43 in the nucleus. gi An ALS motor neuron lacking immunoreactivity to ADAR2 showed piTDP-43 positive cytoplasmic inclusions (arrow) and loss of piTDP-43 immunoreactivity in the nucleus. Asterisks indicate lipofuscin autofluorescence. Bar indicates 20 μm

ADAR2, pTDP-43 and piTDP-43 expression in ALS spinal motor neurons

Both ADAR2-positive (Fig. 4c open arrow) and -negative motor neurons (Fig. 4c closed arrow) were observed in the ALS spinal cords. The immunoreactivity in the ADAR2-positive neurons was observed in the cytoplasm, but apparently not in the nuclei (Figs. 4c open arrow, e, f, 5d, f), as observed in the control motor neurons. These ADAR2-positive neurons showed normal piTDP-43 immunoreactivity in the nucleus (Figs. 4e′, f′, 5e, f,), but exhibited no pTDP-43-positive inclusions (Fig. 4c, c′). In contrast, all ADAR2-negative neurons showed pTDP-43-positive inclusions in the cytoplasm (Fig. 4c′, d′).

Cell count of anterior horn motor neurons

The number of anterior horn cells (motor neurons) (AHC) in 7 sporadic ALS cases was reduced to 39 ± 21% (mean ± SD) of the number in control cases (p < 0.0001, Mann–Whitney’s U test, Fig. 6). A significant proportion of motor neurons (98 out of 170 anterior horn cells; 58%) in the spinal cords obtained from patients with ALS were ADAR2-negative (Table 1; Fig. 6). ADAR2-negative motor neurons were observed in all ALS cases examined, but the proportions varied from 30% in case 3 to 100% in case 2 (Table 1). Notably, all the ADAR2-negative motor neurons had pTDP-43-positive inclusions in the cytoplasm. Conversely, virtually all the ADAR2-positive motor neurons had piTDP-43 immunoreactivity in their nuclei, but did not exhibit pTDP-43-positive cytoplasmic inclusions (Table 1; Fig. 6). Only one motor neuron was stained with both ADAR2 and pTDP-43 (n = 1 in case 4), but none of the motor neurons lacked immunoreactivity to both ADAR2 and pTDP-43. These results indicate a strong association between ADAR2-deficiency and the development of pTDP-43-positive inclusions in the motor neurons of patients with sporadic ALS. However, there is no apparent relationship between the duration of disease and the number of remaining motor neurons with ADAR2 positivity and/or pTDP-43 positivity.
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Fig. 6

Motor neurons with different immunoreactivities in ALS and control cases. The number of anterior horn cells (motor neurons) (AHCs) in 7 sporadic ALS cases was reduced to 39 ± 21% (mean ± SD) of the number in control cases (p < 0.0001, Mann–Whitney U test). In ALS cases, 42% of total AHCs were ADAR2-positive and pTDP-43-negative (pink bar); 58% were ADAR2-negative and pTDP-43-positive (blue bar). Only one AHC out of 170 (0.2%) was positive for both ADAR2 and pTDP-43, and none were negative for both ADAR2 and pTDP-43. All lumbar spinal motor neurons (n = 380) from 6 control cases were ADAR2-positive and pTDP-43-negative (pink bar). AHC anterior horn cell (motor neuron)

Discussion

ADAR2 expression was observed in all 380 spinal motor neurons examined in the control cases in this study, and in a portion of the spinal motor neurons from ALS cases (approximately 42% of 170 neurons). The nuclei of these ADAR2-positive neurons were also immunoreactive for piTDP-43. Notably, more than half the motor neurons in ALS cases lacked immunoreactivity to both ADAR2 and piTDP-43, but these double-negative neurons always displayed pTDP-43-positive inclusions in the cytoplasm. Therefore, normal motor neurons express ADAR2 without forming phosphorylated TDP-43-positive cytoplasmic inclusions, whereas motor neurons lacking ADAR2 in sporadic ALS formed inclusions.

ADAR2 immunoreactivity was exclusively observed in the nuclei of rat motor neurons on the frozen sections created 0–6-h postmortem. Because the density of motor neurons in the spinal ventral gray matter is too low to detect ADAR2 by Western blotting analysis, we used frozen human brain for the analysis, which demonstrated that RED1 specifically recognized two isoforms of active ADAR2 protein in the nuclear fraction [17]. The nuclear localization of the ADAR2 protein in the motor neurons was demonstrated immunohistochemically in frozen human spinal cord sections. In addition, expression of ADAR2 mRNA in the human spinal cord was demonstrated [18]. These results are consistent with the function of ADAR2 in the cell nucleus, which is catalysis of the conversion of adenosine to inosine (A-to-I) at various pre-mRNA positions including the Q/R site of GluR2. We also observed ADAR2 immunoreactivity in the cytoplasm of motor neurons in human paraffin-embedded sections. Importantly, the nuclei of motor neurons were predominantly immunoreactive to ADAR2 in frozen sections of the same control subject. In paraffin-embedded sections and frozen sections from the spinal cord after a 24-h postmortem interval, there was a reduction in ADAR2 immunoreactivity in the nucleus with the concomitant appearance of immunoreactivity in the cytoplasm of rat motor neurons. Therefore, ADAR2 immunoreactivity in the cytoplasm likely represented ADAR2 protein translocated from the nucleus to the cytoplasm resulting from the procedure of paraffin embedding and/or the delay between death and tissue fixation, which are unavoidable during routine neuropathological examinations of human autopsy materials. Because all the control motor neurons demonstrated cytoplasmic ADAR2 immunoreactivity, it is likely that ADAR2-negative motor neurons in ALS spinal cords lacked ADAR2 protein localized to the nucleus.

ADAR2 is involved in the A-to-I conversion of various pre-mRNAs and specifically catalyzes GluR2 Q/R site-editing. AMPA receptors containing GluR2 which is unedited at the Q/R site have significantly higher Ca2+ permeability than those containing edited GluR2. This factor plays a crucial role in neuron survival [5, 31]. Neurons in the mammalian brain only express Q/R site-edited GluR2 mRNA, and mice unable to edit this site die from status epilepticus early in life [14]. GluR2 Q/R site-editing occurs with 100% efficiency in normal human motor neurons, but is characterized by high variability (from 0 to 100%) among individual motor neurons in individual cases of ALS [16]. Therefore, ADAR2-positive motor neurons likely represent normal neurons expressing Q/R site-edited GluR2, whereas ADAR2-negative motor neurons represent those expressing Q/R site-unedited GluR2 [16, 20]. The present results indicating the presence of both ADAR2-positive and -negative motor neurons are consistent with high variability in the efficiency of GluR2 Q/R site-editing (from 0 to 100%) among individual motor neurons in ALS patients [16]. These findings strengthen the hypothesis that reduced ADAR2 activity is closely associated with the pathogenesis of sporadic ALS [20].

The presence or absence of ADAR2 immunoreactivity in the cytoplasm of motor neurons was conversely related to pTDP-43 immunoreactivity in the cytoplasm. Abnormally processed TDP-43 was initially identified as a protein component of ubiquitin-positive and tau-negative inclusions in the brains of patients with FTD and ALS [3, 25]. Subsequently, abnormal TDP-43-positive inclusions were found in various proportions in neurons from patients with other neurodegenerative disorders, such as Parkinson’s disease dementia and dementia with Lewy bodies [24], Parkinsonism-dementia complex and ALS in Guam [8, 11], corticobasal degeneration [36] and Alzheimer’s disease [2, 13, 36]. These results imply that aberrant processing of TDP-43 may be involved in a common pathway of the neurodegenerative process and that the accumulation of pTDP-43 in the cytoplasm of motor neurons is not a disease-specific event in ALS [2, 13, 24, 36].

This study demonstrates that all ADAR2-negative motor neurons showed pTDP-43-positive inclusions in the cytoplasm in cases of sporadic ALS, suggesting a molecular association between reduced ADAR2 activity and the formation of pTDP-43-positive inclusions in ALS motor neurons. Both TDP-43 and ADAR2 are nuclear proteins, playing roles in the regulation of RNA processing; TDP-43 regulates RNA splicing [4, 28] and ADAR2 catalyzes RNA editing. However, there is no report regarding the functional link between the two molecules. We found that pTDP-43-positive inclusions showed no ADAR2 immunoreactivity, indicating that the trapping of ADAR2 protein in the inclusions due to direct protein–protein interaction is unlikely. Reduced ADAR2 activity increases Ca2+ permeable AMPA receptors by failure to edit the Q/R site of GluR2 [5, 6, 14], but it is not known whether an increase of the Ca2+ overload influences the TDP-43 processing. Thus, it is not clear from the present immunohistochemical study whether the reduced ADAR2 expression is a cause or a consequence of TDP-43 pathology. Interestingly, neither pTDP-43-positive inclusions [23, 35] nor a reduction of GluR2 Q/R-site-editing [19] was associated with SOD1-related familial ALS or SBMA, an X-linked hereditary lower motor neuron disease associated with expanded CAG repeats in the androgen receptor gene. Consistent with the absence of pTDP-43-positive inclusions in the spinal motor neurons of SOD1-associated familial ALS, present study demonstrated that all the motor neurons examined were ADAR2-positive in SOD1G93A transgenic mouse spinal cords. Elucidation of the molecular mechanism underlying the co-occurrence of reduced ADAR2 activity and abnormal TDP-43 pathology in the same motor neurons may provide a clue to the neurodegenerative process of sporadic ALS.

Supplementary material

401_2010_678_MOESM1_ESM.ppt (2.2 mb)
Supplementary Figure 1ADAR2 immunostaining in degenerating neurons in other neurological diseases. Neurons in the pontine nuclei of an ALS patient exhibit slight ADAR2 immunoreactivity in the cytoplasm (a). The neurons in the pontine nuclei in both multiple system atrophy (b) and spinocerebellar atrophy type 1 (c) showed faint ADAR2 immunoreactivity, although these neurons were atrophic and reduced in number. These results suggested that the alteration of ADAR2 activity was not involved in the process of neuronal death in the pontine nucleus of MSA or SCA1. Bar indicates 20 μm (PPT 2238 kb)
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Supplementary Figure 2Immunohistochemistry with two anti-ADAR2 antibodies. Both RED1 (a) and C-15 (b) stained specifically the cytoplasm but not the nucleus of motor neurons. Non-specific lipofuscin staining is observed in (b) (PPT 1858 kb)
401_2010_678_MOESM3_ESM.doc (24 kb)
Supplementary material 3 (DOC 24 kb)

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