- Open Access
Heterogeneity of cerebral TDP-43 pathology in sporadic amyotrophic lateral sclerosis: Evidence for clinico-pathologic subtypes
- Ryoko Takeuchi†1, 2,
- Mari Tada†1,
- Atsushi Shiga†3,
- Yasuko Toyoshima1,
- Takuya Konno2,
- Tomoe Sato1, 2,
- Hiroaki Nozaki2,
- Taisuke Kato3,
- Masao Horie4,
- Hiroshi Shimizu1,
- Hirohide Takebayashi4,
- Osamu Onodera2,
- Masatoyo Nishizawa2,
- Akiyoshi Kakita1 and
- Hitoshi Takahashi1Email author
© The Author(s). 2016
Received: 11 June 2016
Accepted: 11 June 2016
Published: 23 June 2016
Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are types of major TDP-43 (43-kDa TAR DNA-binding protein) proteinopathy. Cortical TDP-43 pathology has been analyzed in detail in cases of FTLD-TDP, but is still unclear in cases of ALS. We attempted to clarify the cortical and subcortical TDP-43 pathology in Japanese cases of sporadic ALS (n = 96) using an antibody specific to phosphorylated TDP-43 (pTDP-43). The cases were divided into two groups: those without pTDP-43-positive neuronal cytoplasmic inclusions in the hippocampal dentate granule cells (Type 1, n = 63), and those with such inclusions (Type 2, n = 33). Furthermore, the Type 2 cases were divided into two subgroups based on semi-quantitative estimation of pTDP-43-positive dystrophic neurites (DNs) in the temporal neocortex: Type 2a (accompanied by no or few DNs, n = 22) and Type 2b (accompanied by abundant DNs, n = 11). Clinico-pathologic analysis revealed that cognitive impairment was a feature in patients with Type 2a and Type 2b, but not in those with Type 1, and that importantly, Type 2b is a distinct subtype characterized by a poor prognosis despite the less severe loss of lower motor neurons, the unusual subcortical dendrospinal pTDP-43 pathology, and more prominent glial involvement in cortical pTDP-43 pathology than other two groups. Considering the patient survival time and severity of motor neuron loss in each group, transition from Type 1 to Type 2, or from Type 2a to Type 2b during the disease course appeared unlikely. Therefore, each of these three groups was regarded as an independent subtype.
Amyotrophic lateral sclerosis (ALS), an adult-onset, fatal neurodegenerative disorder, is the most common type of motor neuron disease (MND). Most cases (90-95 %) appear to occur randomly without a family history (sporadic ALS) [1, 2]. The principal feature is progressive muscular weakness due to degeneration of both the upper and lower motor neuron systems, and characteristic ubiquitin-positive neuronal cytoplasmic inclusions (NCIs) are present in the lower motor neurons [3–5]. It has also been well recognized that there are sporadic ALS cases accompanied by cognitive impairment (ALS or MND with dementia: ALS-D or MND-D), in which the presence of ubiquitin-positive NCIs and dystrophic neurites (DNs) in the extra-motor cortices, including the hippocampal dentate gyrus, is a significant feature [5–9]. Similar ubiquitin pathology has also been demonstrated in a subset of patients with frontotemporal dementia (FTD) with or without MND . Accordingly, it has been suggested that ALS, ALS-D (MND-D) and FTD without MND represent a clinico-pathologic spectrum .
Since the identification of a nuclear protein, 43-kDa TAR DNA-binding protein (TDP-43, also known as TARDBP), as the major component of the ubiquitinated inclusions in frontotemporal lobar degeneration with ubiquitin-positive, tau-and α-synuclein-negative inclusions (FTLD-U) and ALS [11, 12], many studies of such cases employing TDP-43 immunohistochemistry have been performed [13–18]. This has led to the recognition that the clinico-pathologic spectrum encompassing ALS at one end and FTD at the other represents a new concept of TDP-43 proteinopathy [19, 20].
Cases of FTLD-U were originally divided into three types by two independent groups using their own classification systems based on the morphology and anatomical distribution of cortical ubiquitin neuronal lesions, including NCIs and DNs [21, 22]. After confirmation that the majority of FTLD-U cases in fact represent FTLD-TDP, these two classification systems were integrated into a ‘harmonized classification system’ that included four types (Types A, B, C and D) of FTLD-TDP pathology , the new Type D being associated with mutations of the VCP (valosin-containing protein) gene [24, 25]. In this new classification system, MND with FTD (so-called ALS-D) was regarded as a common phenotype of Type B, being characterized by moderate numbers of NCIs and very few DNs throughout all cortical layers. However, one of the above studies demonstrated that 7 of 10 cases that were sporadic and exhibited MND in addition to dementia had cortical ubiquitin pathology characterized by a mixture of numerous NCIs and frequent small DNs , corresponding to Type A of the new classification system mentioned above ; this finding suggested that the cortical ubiquitin pathology in ALS-D and/or FTLD-MND could be heterogeneous.
Using a phosphorylation-independent anti-TDP-43 antibody, we have previously demonstrated that immunopositive NCIs and glial cytoplasmic inclusions (GCIs) can occur in many brain regions in ALS, and that cases can be classified into two types – type 1 and type 2–based on the distribution pattern of NCIs in the CNS and hierarchical cluster analysis of the pattern . Type 2 can be distinguished from type 1 by the presence of TDP-43-positive NCIs in the extra-motor neuron system, including the frontotemporal cortex, hippocampal formation, neostriatum and substantia nigra, and is significantly associated with dementia . Since a monoclonal antibody specifically recognizing abnormally phosphorylated TDP-43 has become available, we have often noticed the presence of abundant threads, or dot-like or granular DNs in the temporal neocortex in cases of ALS, more strictly those with NCIs in the hippocampal dentate granule cells.
In the present study, we attempted to reevaluate the cortical and subcortical TDP-43 pathology in cases of sporadic ALS using the above monoclonal antibody, which never recognizes endogenous non-phosphorylated TDP-43 in nuclei, thus allowing unambiguous identification of pathologic structures. The results obtained eventually allowed us to classify the examined cases into three pathologic groups, whose clinical, pathologic and biochemical features were then analyzed.
Materials and methods
The present study was conducted within the framework of a project, “Neuropathologic and Molecular-Genetic Investigation of CNS Degenerative Diseases”, approved by the Institutional Review Board of Niigata University. Informed consent was obtained from the patients’ families prior to genetic analyses.
We retrieved all cases of pathologically confirmed ALS from our institutional autopsy files covering the period between 1975 and 2013, reviewed the medical records and identified 128 cases of clinically sporadic ALS without any family histories of similar neurological disorders. All of the patients were of Japanese ancestry, and their clinical information was obtained retrospectively by reviewing their medical records.
Among these 128 cases, the tissue samples were of poor quality due to complications of infarction, etc. and/or sampling in 26 cases, pathologic features indicative of complications arising from other major neurodegenerative diseases affecting the cerebral cortex and basal ganglia were evident in 4 cases (Alzheimer’s disease = 2; progressive supranuclear palsy = 1; multiple system atrophy = 1), and no TDP-43-positive inclusions were detected in the CNS, including the lower motor neurons, in 2 cases. Accordingly, a total of 32 cases were excluded, leaving 96 cases (58 male, 38 female; mean age 67.4 years, standard deviation 9.8 years, range 36–87 years) for analysis. Seven cases were found to have only a few Lewy bodies, with α-synuclein-positive NCIs and DNs confined to the brainstem. These cases were considered to be incidental Parkinson’s disease and were included in the present study. All of the studied cases showed loss of upper and lower motor neurons as well as ubiquitin-positive skein-like inclusions in the remaining lower motor neurons, and Bunina bodies were evident in the remaining lower motor neurons in 91 of the 96 cases.
Histology and immunohistochemistry
Multiple formalin-fixed, paraffin-embedded CNS tissue blocks for all cases were available for the present study. For the motor cortex, frontal cortex (including the prefrontal area), temporal cortex (including the hippocampus), basal ganglia, hypoglossal nucleus, and cervical and lumbar anterior horns, 4-μm-thick sections stained with hematoxylin-eosin (H-E) were used for semi-quantitative analysis employing a 4-point scale (0, absent; 1, mild; 2, moderate; 3, severe) of neuronal cell loss (Additional file 1: Figure S1). FTLD was diagnosed by the presence of atrophy and neuronal loss with gliosis in the frontotemporal cortices, regardless of severity. The study was carried out by two of the authors (R.T. and M.T.), and reviewed by two other investigators (Y.T. and H.T.) to ensure evaluation consistency.
Newly prepared 4-μm-thick sections were cut from the temporal cortex (including the hippocampus), frontal and motor cortices and basal ganglia for immunohistochemical studies. The sections were autoclaved at 120 °C in 10 mM citrate buffer, pH 6.0, for 10 min, and then immunostained with a mouse monoclonal antibody against phosphorylated TDP-43 (pTDP-43; phospho Ser409/410) (clone 11–9; Cosmo Bio Co., Ltd., Tokyo, Japan; 1:5000). Selected sections were also immunostained with a rabbit polyclonal phosphorylation-independent anti-TDP-43 antibody (10782-2-AP; Protein Tech Group Inc., Chicago, IL; 1:4000). Immunolabeling was detected using the peroxidase-polymer-based method using a Histofine Simple Stain MAX-PO kit (Nichirei Biosciences Inc, Tokyo, Japan) with diaminobenzidine (DAB) as the chromogen. To estimate the neuropathological staging of changes associated with Alzheimer’s disease, we performed Gallyas-Braak silver impregnation, and immunohistochemistry using mouse monoclonal antibodies against hyperphosphorylated tau protein (AT8; Innogenetics, Ghent, Belgium; 1:200) and β-amyloid (Dako, Glostrup, Denmark; 1:100). Then, we evaluated the Braak stages of neurofibrillary tangles and amyloid deposits [26, 27], and also estimated the level of Alzheimer’s disease-related neuropathologic change based on ‘ABC’ score [26–30].
Classification procedure based on cortical pTDP-43 pathology
A double-labeling immunofluorescence study was performed to assess the anatomical localization of pTDP-43 deposits forming granular and dot-like DNs. Sections of the temporal lobe and basal ganglia, including the neostriatum and globus pallidus from three representative cases of Type 2b were examined using rabbit polyclonal anti-pTDP-43 (phospho Ser409/410) (Cosmo Bio Co., Ltd.; 1:2000) and mouse monoclonal anti-neurofilament H (non-phosphorylated) (SMI-32; Calbiochem, San Diego, CA; 1:500), as well as rabbit polyclonal anti-pTDP-43 (phospho Ser409/410) and mouse monoclonal anti-synaptophysin (Leica Biosystems; Newcastle-upon-Tyne, UK; 1:50). The second antibodies used were Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 555 goat anti-mouse IgG (Molecular Probes, Eugene, OR; 1:1000). The sections were treated with an Autofluorescence Eliminator Reagent (Millipore, Billerica, MA), mounted under glass coverslips using VectaShield mounting medium with 4,6-diamidino-2-phenylindole (DAPI) nuclear stain (Vector Laboratories, Burlingame, CA), and analyzed using a Carl Zeiss confocal laser scanning microscope (LSM700).
Double labeling with in situ hybridization and immunohistochemistry
The numbers of neurons and glial cells possessing cytoplasmic pTDP-43-positive inclusions (NCIs and GCIs) were assessed in the motor cortex using a double-labeling method with in situ hybridization (ISH) and immunohistochemistry, and compared between the three groups. For this study, we selected representative cases of Type 1 (n = 22), Type 2a (n = 12) and Type 2b (n = 7) among cases logged after 1990. In the Type 1 cases, a significant number of pTDP-43-positive NCIs and GCIs were seen in the motor cortex. Therefore, from the Type 2a and Type 2b cases, we selected cases in which a larger number of pTDP-43-positive NCIs and GCIs were evident in the motor cortex than in the temporal or frontal cortex.
ISH was performed in these three groups using newly prepared paraffin-embedded 10-μm-thick sections from the motor cortex, as described previously , with minor modifications (Supplementary Methods). A probe for human neurofilament 3 (hNF: 150-kDa medium) (GenBank accession number: BC002421) was used. As a result, the sections from 11 cases were found to be inadequate for ISH, leaving 30 cases (Type 1, n = 16; Type 2a, n = 8; Type 2b, n = 6) logged between 1990 and 2012 for the subsequent immunohistochemical study.
The hybridized sections were immunostained using mouse monoclonal anti-pTDP-43 (clone 11–9; 1:5000), and then counterstained with Nuclear Fast Red solution (Sigma Aldrich, St. Louis, MO). In each case, 10 sequential images of the motor cortex were taken through a × 20 objective lens using a single ISH-labeled and immunostained section. The total area taken from a section from each case was 1.5 mm2, covering the cortical layers II-VI. The numbers of hNF-positive cells with nuclei, hNF- and pTDP-43-positive cells with nuclei, and hNF-negative and pTDP-43-positive cells with nuclei were counted manually.
To compare clinical and pathologic features between the three groups, we used Kruskal-Wallis test with post-hoc Steel-Dwass test for non-parametric analysis of independent samples, multiple regression analysis to determine whether independent variables can predict the value of the dependent variable, Kaplan-Meier plots and a log-rank test to compare survival distributions, and Fisher’s exact test with Bonferroni-corrected multiple comparisons or Ryan’s multiple comparison test for comparison of categorical data. These statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA), SPSS Statistics version 12.0 (IBM, Armonk, NY) and R version 3.1.2 (http://www.r-project.org/). Differences were considered statistically significant at P <0.05.
Biochemical analysis of pTDP-43
Fractionation of frozen brain tissues and TDP-43 immunoblotting were performed on selected cases. Protein lysates were generated from the motor cortex of Type 1 (n = 4) cases and from the temporal cortex of Type 2a (n = 4) and Type 2b (n = 4) cases, as well as from the frontal cortex of FTLD-TDP Type A, B and C (two each), as described previously [32–34], with minor modifications (Additional file 2). In order to distinguish the 20-25-kDa band pattern clearly, we used a large polyacrylamide gel (184 × 185 mm) and electrophoresed the samples at 200 V for 16 h at 4 °C. The separated samples were analyzed by immunoblotting with a mouse monoclonal antibody against pTDP-43 (clone 11–9; 1:2000) .
Analysis of the TARDBP and C9ORF72 genes
The presence or absence of TARDBP and C9ORF72 gene mutations was analyzed in cases for which frozen tissue samples were available. High-molecular-weight genomic DNA was extracted from 83 cases (Type 1, n = 54; Type 2a, n = 19; Type 2b, n = 10). We amplified all the exons of the TARDBP (NM_007375.3) gene using a series of primers, followed by sequence reaction . For screening of GGGGCC repeat expansion in C9ORF72 (NM_018325.2), repeat-primed PCR was performed on an ABI 3130xl genetic analyzer (Applied Biosystems, Foster City, CA) using Peak Scanner software v1.0 (Applied Biosystems), as described previously [37, 38].
Demographics and clinical features in three types of sporadic ALS
(n = 96)
(n = 63)
(n = 22)
(n = 11)
Sex (male: female)
Age at onseta (years)
Survival timea (months)
61 (64 %)
50 (79 %)***
7 (32 %)
4 (36 %)
32 (33 %)
13 (21 %)
13 (59 %)
6 (55 %)
3 (3 %)
2 (9 %)
1 (9 %)
15 (16 %)
1 (2 %)****
7 (32 %)
7 (64 %)
Cause of death
67 (70 %)
45 (71 %)
14 (64 %)
8 (73 %)
29 (30 %)
18 (29 %)
8 (36 %)
3 (27 %)
Pathologic features and phosphorylated TDP-43 pathology in three types of sporadic ALS
(n = 63)
(n = 22)
(n = 11)
2.1 ± 0.7
2.1 ± 0.7
2.5 ± 0.7
Anterior horns (cervical and lumbar)
2.2 ± 0.5
2.1 ± 0.6
1.5 ± 0.4*
2.3 ± 0.7
2.1 ± 0.7
1.4 ± 0.5**
Frontotemporal degeneration (cases)
4 (6 %)***
8 (36 %)
7 (64 %)
pTDP-43 pathology (cases)
NCIs: temporal-predominant type
13 (59 %)
5 (45 %)
DNs: many in neostriatum and globus pallidus
1 (2 %)
2 (9 %)
9 (82 %)****
In each group, a proportion of cases showed FTLD manifested by frontotemporal atrophy and frontotemporal cortical neuronal loss with gliosis; the rate of occurrence of FTLD was only 6 % in Type 1, being much lower than in Type 2a (36 %, P <0.001) or in Type 2b (64 %, P = 0.00001). In around half of the cases in both the Type 2a and Type 2b groups, pTDP-43-positive NCIs showed a temporal cortex-predominant distribution pattern, in comparison with the motor cortex. No pTDP-43-positive neuronal intranuclear inclusions were evident in any of the cases examined.
The anatomical localization of pTDP-43 deposits forming granular and dot-like DNs
pTDP-43 inclusion-bearing neurons and oligodendrocytes in the motor cortex
The density of NCI-bearing neurons was higher in Type 2b than in Type 1 (P = 0.032) (Fig. 4b i), although there was no difference in the total number of neurons between the two; the median neuron count per 1 mm2 was 553.8 (interquartile range: 503.7-577.8) for Type 1, 573.7 (516.7-619.7) for Type 2a, and 575.4 (533.3-650.3) for Type 2b, P = 0.316. The density of GCI-bearing glial cells was higher in Type 2b than that in Type 1 (P = 0.027) and Type 2a (P = 0.018) (Fig. 4b ii). The ratio of NCI-bearing neurons to total NCI-bearing neurons and GCI-bearing glial cells was higher in Type 2a than in Type 1 (P = 0.004) (Fig. 4b iii), although there was no difference in the density of NCI-bearing neurons between the two (P = 0.114) (Fig. 4b i).
Biochemical and genetic features
With regard to the sequencing of the TDP-43 (TARDBP) gene in the 83 cases examined, there were no mutations in the coding regions except for one case with a known synonymous mutation in exon 6 . No C9ORF72 repeat expansion was observed.
In the present study, a series of 96 cases of sporadic ALS were classified into three groups on the basis of the cortical pTDP-43 pathology (Fig. 1a). Based on the absence or presence of pTDP-43-positive NCIs in the hippocampal dentate granule cells, we divided all cases into two groups, Type 1 and Type 2 (Fig. 1a, b). Among the cases belonging to Type 2, we noted that some were characterized by the presence of DNs in the affected temporal neocortex in addition to NCIs (Fig. 1a, c). Accordingly, we further divided these Type 2 cases into two subgroups, i.e. Type 2a and Type 2b, based on the presence or absence of the prominent appearance of pTDP-43-positive DNs, irrespective of the amount of NCI-bearing neurons. Interestingly, statistical analysis revealed a number of significant differences of clinico-pathologic features among these three groups, which appeared to support the validity of this classification (Tables 1, 2).
Comparison of the ubiquitin- or TDP-43-related cortical pathology of FTLD described by many investigators suggested that Type 2a apparently corresponded to Type B in the harmonized classification system for FTLD-TDP pathology reported by Mackenzie et al. (2011) [16, 21, 22, 40, 42], whereas Type 2b showed a very unusual cortical TDP-43 pathology characterized by many DNs in the form of threads, and granular and dot-like structures (Fig. 1c). This pathologic picture appeared to be somewhat similar to, but by no means consistent with that of Type A in the classification system mentioned above (the majority of DNs observed in cases classified as Type 2b appeared to be smaller structures). Furthermore, immunoblot analysis of sarkosyl-insoluble pTDP-43 revealed that the band patterns of Type 2b and Type 1 were indistinguishable and differed from those of FTLD-TDP Type A (Fig. 5). By contrast, Type A pathology and a FTLD-TDP Type A band pattern have been described in the motor cortex in two cases of primary lateral sclerosis (PLS) . Therefore, we conclude that, to our knowledge, the overall picture of Type 2b is distinct from those observed in patients with FTLD-U or FTLD-TDP.
From a clinico-pathologic viewpoint, cognitive impairment was much less frequent in patients with Type 1 as we suggested previously ; in other words, its presence in patients with sporadic ALS strongly suggests Type 2a or Type 2b. Moreover, Type 2b, although accounting for only a small proportion of the disease spectrum, could be regarded as a distinct subset showing a relatively old age at onset and a shorter survival time than the other two groups. Patients in this group frequently developed bulbar symptoms initially (Table 1). From a pathologic viewpoint, less severe loss of lower motor neurons and the presence of numerous pTDP-43-positive granular and dot-like DNs in the putamen and globus pallidus were notable features in Type 2b (Table 2).
In the present study, clinical information was limited to that obtained retrospectively from the medical records of the patients for whom details of some clinical symptoms were unavailable. This was one of the limitations of the present study. It has been shown that classification of patients with ALS based on clinical assessment provides information for better understanding the wide spectrum of this motor and cognitive disorder . Moreover, neuropathologic examination of a large-scale cohort of clinically assessed patients would be expected to reveal pathognomonic features responsible for the various symptoms.
The results of double-labeling immunofluorescence using anti-pTDP-43 antibody and dendritic (SMI-32) or presynaptic (synaptophysin) markers in the temporal cortex and basal ganglia of patients with Type 2b suggested that deposition of pTDP-43 might occur in neuronal dendritic spines (Fig. 3). TDP-43 is normally localized primarily to the nucleus, where it regulates transcription and alternative splicing [12, 32]. In addition, recently, the roles of TDP-43 in dendrites and spines have been successively investigated [44, 45]. In neurons, TDP-43 is expressed abundantly in dendrites mainly in the form of RNA granules, and translocation of the TDP-43-containing granules into dendritic spines in response to neuronal activities has been demonstrated in cultured neurons. Accordingly it has been proposed that TDP-43 is a neuronal activity-responsive factor functioning in the regulation of neuronal plasticity . More recently, it has been reported that disease-linked mutations in TDP-43 alter the dynamics of neuronal RNA granules during dendritic arborization, and reduce or slow the response of TDP-43 to changes in neuronal activity . Considering these findings, it could be speculated that accumulation of the pathologic protein, pTDP-43, in neuronal dendritic spines of the cerebral cortex, as well as in the neostriatum and globus pallidus, leads to a decline in synaptic plasticity, and is at least partly responsible for the poor prognosis of patients with Type 2b, mainly through respiratory failure, despite the fact that loss of lower motor neurons is milder than that seen in patients with Type 1 and Type 2a.
Comparison of the TDP-43 cellular pathology in the motor cortex among the three disease groups showed that both the density of NCI-bearing neurons and GCI-bearing glial cells were higher in Type 2b than in Type 1. In addition, the ratio of NCI-bearing neurons to all inclusion-bearing cells was higher in Type 2a than in Type 1. Propagation of pathologic TDP-43 via axonal connections has been proposed on the basis of findings from autopsied ALS brains , and it has also been demonstrated that oligodendroglial involvement by pathologic TDP-43 precedes neuronal involvement in the spinal cord in ALS . However, the mechanisms responsible for the spread of TDP-43 pathology into multiple systems of the CNS remain uncertain. Our present findings indicate that the mechanism of cellular involvement in propagation differs between Type 2b and Type 2a, oligodendrocytes also being substantially involved, in addition to neurons, in the former.
We consider that the three groups of sporadic ALS described here are originally independent of each other. Based on the TDP-43 pathologic staging scheme proposed by Brettschneider et al. , all cases of Type 2 in the present study corresponded to Stage 4, and the TDP-43 pathology appeared to progress from Stage 1 to 4 during the disease course. However, in sporadic ALS, we have previously demonstrated that long-term survival facilitated by artificial respiratory support has no apparent influence on the distribution pattern of neuronal inclusions in the two types (type 1 and type 2) of TDP-43 , and that 5 of 6 patients who survived for 10–20 years without respiratory support showed type 1 TDP-43 pathology . Moreover, the present study revealed that survival time was shorter and that loss of lower motor neurons was less severe in patients with Type 2b than in those suffering from Type 1 and Type 2a. Taken together, we concluded that transition from Type 1 to Type 2, or from Type 2a to Type 2b, depending on disease duration or progression, would be unlikely to occur.
None of the patients in the present study had mutations in C9ORF72. Hexanucleotide (GGGGCC) repeat expansion in a non-coding region of C9ORF72 is the major genetic cause of FTD and ALS (c9FTD/ALS) in the Caucasian population , but extremely rare in Japan [38, 50, 51]. When considering the rarity of this genetic disease in Japan, it is interesting to note that the reported national incidence rate of ALS in 2009 was 2.3 per 100,000 population , being much lower than the rates reported for Caucasian populations in Europe and North America .
In conclusion, we have demonstrated heterogeneity of cortical pTDP-43 pathology in sporadic ALS, allowing the disease to be classified into three groups: Type 1, and Type 2a and Type 2b. Several significant clinical and pathologic correlations among these groups were evident. It was re-confirmed that dementia is not a feature of Type 1. Furthermore, the distinct subtype, Type 2b, accompanied by dendrospinal accumulation of pTDP-43, was shown to be characterized by a poor prognosis despite the less severe loss of lower motor neurons, the unusual subcortical dendritic pTDP-43 pathology, and more prominent glial involvement in cortical pTDP-43 pathology than other two groups. Further studies will be needed in order to establish the character of this subtype, Type 2b. Recognition of the heterogeneity of the anatomical and cellular localization of pTDP-43 will be important for clarifying the pathomechanisms involved in pTDP-43 aggregation and propagation in sporadic ALS, and eventually for developing future strategies of treatment and prevention.
ALS, amyotrophic lateral sclerosis; DN, dystrophic neurite; FTLD, frontotemporal lobar degeneration; GCI, glial cytoplasmic inclusion; MND, motor neuron disease; NCI, neuronal cytoplasmic inclusion; pTDP-43, phosphorylated 43-kDa TAR DNA-binding protein
The authors are very grateful to Prof. Kouhei Akazawa, and Drs. Akio Yokoseki and Yuichi Yokoyama for advice on statistical analysis, and also thank C. Tanda, S. Nigorikawa, J. Takasaki, H. Saito, T. Fujita and S. Hirokawa for their technical assistance, and M. Machida and Y. Ueda for secretarial assistance.
This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (26640029 and 26250017 to H. T.), and a grant from the Ministry of Health, Labour and Welfare of Japan (to H. T.).
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- Renton AE, Chiò A, Traynor BJ. State of play in amyotrophic lateral sclerosis genetics. Nat Neurosci. 2014;17:17–23. doi:10.1038/nn.3584.View ArticlePubMedGoogle Scholar
- Turner MR, Hardiman O, Benatar M, Brooks BR, Chio A, De Carvalho M, Ince PG, Lin C, Miller RG, Mitsumoto H, Nicholson G, Ravits J, Shaw PJ, Swash M, Talbot K, Traynor BJ, Van den Berg LH, Veldink JH, Vucic S, Kiernan MC. Controversies and priorities in amyotrophic lateral sclerosis. Lancet Neurol. 2013;12:310–22. doi:10.1016/S1474-4422(13)70036-X.View ArticlePubMedPubMed CentralGoogle Scholar
- Leigh PN, Anderton BH, Dodson A, Gallo JM, Swash M, Power DM. Ubiquitin deposits in anterior horn cells in motor neurone disease. Neurosci Lett. 1988;93:197–203. doi:10.1016/0304-3940(88)90081-X.View ArticlePubMedGoogle Scholar
- Lowe J, Lennox G, Jefferson D, Morrell K, McQuire D, Gray T, Landon M, Doherty FJ, Mayer RJ. A filamentous inclusion body within anterior horn neurones in motor neurone disease defined by immunocytochemical localisation of ubiquitin. Neurosci Lett. 1988;94:203–10. doi:10.1016/0304-3940(88)90296-0.View ArticlePubMedGoogle Scholar
- Piao YS, Wakabayashi K, Kakita A, Yamada M, Hayashi S, Morita T, Ikuta F, Oyanagi K, Takahashi H. Neuropathology with clinical correlations of sporadic amyotrophic lateral sclerosis: 102 autopsy cases examined between 1962 and 2000. Brain Pathol. 2003;13:10–22.View ArticlePubMedGoogle Scholar
- Mackenzie IR, Feldman HH. Ubiquitin immunohistochemistry suggests classic motor neuron disease, motor neuron disease with dementia, and frontotemporal dementia of the motor neuron disease type represent a clinico-pathologic spectrum. J Neuropathol Exp Neurol. 2005;64:730–9.View ArticlePubMedGoogle Scholar
- Nakano I. Frontotemporal dementia with motor neuron disease (amyotrophic lateral sclerosis with dementia). Neuropathology. 2000;20:68–75. doi:10.1046/j.1440-1789.2000.00272.x.View ArticlePubMedGoogle Scholar
- Okamoto K, Hirai S, Yamazaki T, Sun XY, Nakazato Y. New ubiquitin-positive intraneuronal inclusions in the extra-motor cortices in patients with amyotrophic lateral sclerosis. Neurosci Lett. 1991;129:233–6. doi:10.1016/0304-3940(91)90469-A.View ArticlePubMedGoogle Scholar
- Okamoto K, Murakami N, Kusaka H, Yoshida M, Hashizume Y, Nakazato Y, Matsubara E, Hirai S. Ubiquitin-positive intraneuronal inclusions in the extramotor cortices of presenile dementia patients with motor neuron disease. J Neurol. 1992;239:426–30. doi:10.1007/BF00856806.View ArticlePubMedGoogle Scholar
- McKhann GM, Albert MS, Grossman M, Miller B, Dickson D, Trojanowski JQ. Clinical and Pathological Diagnosis of Frontotemporal Dementia: report of the Work Group on Frontotemporal Dementia and Pick's Disease. Arch Neurol. 2001;58:1803–9. doi:10.1001/archneur.58.11.1803.View ArticlePubMedGoogle Scholar
- Arai T, Hasegawa M, Akiyama H, Ikeda K, Nonaka T, Mori H, Mann D, Tsuchiya K, Yoshida M, Hashizume Y, Oda T. TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem Biophys Res Commun. 2006;351:602–11. doi:10.1016/j.bbrc.2006.10.093.View ArticlePubMedGoogle Scholar
- Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, Bruce J, Schuck T, Grossman M, Clark CM, McCluskey LF, Miller BL, Masliah E, Mackenzie IR, Feldman H, Feiden W, Kretzschmar HA, Trojanowski JQ, Lee VM. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. 2006;314:130–3. doi:10.1126/science.1134108.View ArticlePubMedGoogle Scholar
- Brandmeir NJ, Geser F, Kwong LK, Zimmerman E, Qian J, Lee VM, Trojanowski JQ. Severe subcortical TDP-43 pathology in sporadic frontotemporal lobar degeneration with motor neuron disease. Acta Neuropathol. 2008;115:123–31. doi:10.1007/s00401-007-0315-5.View ArticlePubMedGoogle Scholar
- Davidson Y, Kelley T, Mackenzie IR, Pickering-Brown S, Du Plessis D, Neary D, Snowden JS, Mann DM. Ubiquitinated pathological lesions in frontotemporal lobar degeneration contain the TAR DNA-binding protein, TDP-43. Acta Neuropathol. 2007;113:521–33. doi:10.1007/s00401-006-0189-y.View ArticlePubMedGoogle Scholar
- Dickson DW. TDP-43 immunoreactivity in neurodegenerative disorders: disease versus mechanism specificity. Acta Neuropathol. 2008;115:147–9. doi:10.1007/s00401-007-0323-5.View ArticlePubMedGoogle Scholar
- Geser F, Martinez-Lage M, Robinson J, Uryu K, Neumann M, Brandmeir NJ, Xie SX, Kwong LK, Elman L, McCluskey L, Clark CM, Malunda J, Miller BL, Zimmerman EA, Qian J, Van Deerlin V, Grossman M, Lee VM, Trojanowski JQ. Clinical and pathological continuum of multisystem TDP-43 proteinopathies. Arch Neurol. 2009;66:180–9. doi:10.1001/archneurol.2008.558.View ArticlePubMedPubMed CentralGoogle Scholar
- Nishihira Y, Tan CF, Onodera O, Toyoshima Y, Yamada M, Morita T, Nishizawa M, Kakita A, Takahashi H. Sporadic amyotrophic lateral sclerosis: two pathological patterns shown by analysis of distribution of TDP-43-immunoreactive neuronal and glial cytoplasmic inclusions. Acta Neuropathol. 2008;116:169–82. doi:10.1007/s00401-008-0385-z.View ArticlePubMedGoogle Scholar
- Tan CF, Eguchi H, Tagawa A, Onodera O, Iwasaki T, Tsujino A, Nishizawa M, Kakita A, Takahashi H. TDP-43 immunoreactivity in neuronal inclusions in familial amyotrophic lateral sclerosis with or without SOD1 gene mutation. Acta Neuropathol. 2007;113:535–42. doi:10.1007/s00401-007-0206-9.View ArticlePubMedGoogle Scholar
- Geser F, Lee VM, Trojanowski JQ. Amyotrophic lateral sclerosis and frontotemporal lobar degeneration: a spectrum of TDP-43 proteinopathies. Neuropathology. 2010;30:103–12. doi:10.1111/j.1440-1789.2009.01091.x.View ArticlePubMedPubMed CentralGoogle Scholar
- Geser F, Martinez-Lage M, Kwong LK, Lee VM, Trojanowski JQ. Amyotrophic lateral sclerosis, frontotemporal dementia and beyond: the TDP-43 diseases. J Neurol. 2009;256:1205–14. doi:10.1007/s00415-009-5069-7.View ArticlePubMedPubMed CentralGoogle Scholar
- Mackenzie IR, Baborie A, Pickering-Brown S, Du Plessis D, Jaros E, Perry RH, Neary D, Snowden JS, Mann DM. Heterogeneity of ubiquitin pathology in frontotemporal lobar degeneration: classification and relation to clinical phenotype. Acta Neuropathol. 2006;112:539–49. doi:10.1007/s00401-006-0138-9.View ArticlePubMedPubMed CentralGoogle Scholar
- Sampathu DM, Neumann M, Kwong LK, Chou TT, Micsenyi M, Truax A, Bruce J, Grossman M, Trojanowski JQ, Lee VM. Pathological heterogeneity of frontotemporal lobar degeneration with ubiquitin-positive inclusions delineated by ubiquitin immunohistochemistry and novel monoclonal antibodies. Am J Pathol. 2006;169:1343–52. doi:10.2353/ajpath.2006.060438.View ArticlePubMedPubMed CentralGoogle Scholar
- Mackenzie IR, Neumann M, Baborie A, Sampathu DM, Du Plessis D, Jaros E, Perry RH, Trojanowski JQ, Mann DM, Lee VM. A harmonized classification system for FTLD-TDP pathology. Acta Neuropathol. 2011;122:111–3. doi:10.1007/s00401-011-0845-8.View ArticlePubMedPubMed CentralGoogle Scholar
- Forman MS, Mackenzie IR, Cairns NJ, Swanson E, Boyer PJ, Drachman DA, Jhaveri BS, Karlawish JH, Pestronk A, Smith TW, Tu PH, Watts GD, Markesbery WR, Smith CD, Kimonis VE. Novel ubiquitin neuropathology in frontotemporal dementia with valosin-containing protein gene mutations. J Neuropathol Exp Neurol. 2006;65:571–81.View ArticlePubMedGoogle Scholar
- Neumann M, Mackenzie IR, Cairns NJ, Boyer PJ, Markesbery WR, Smith CD, Taylor JP, Kretzschmar HA, Kimonis VE, Forman MS. TDP-43 in the ubiquitin pathology of frontotemporal dementia with VCP gene mutations. J Neuropathol Exp Neurol. 2007;66:152–7.View ArticlePubMedGoogle Scholar
- Braak H, Alafuzoff I, Arzberger T, Kretzschmar H, Del Tredici K. Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry. Acta Neuropathol. 2006;112:389–404.View ArticlePubMedPubMed CentralGoogle Scholar
- Braak H, Braak E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol. 1991;82:239–59.View ArticlePubMedGoogle Scholar
- Mirra SS, Heyman A, McKeel D, Sumi SM, Crain BJ, Brownlee LM, Vogel FS, Hughes JP, Van Belle G, Berg L. The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer’s disease. Neurology. 1991;41:479–86.View ArticlePubMedGoogle Scholar
- Montine TJ, Phelps CH, Beach TG, Bigio EH, Cairns NJ, Dickson DW, Duyckaerts C, Frosch MP, Masliah E, Mirra SS, Nelson PT, Schneider JA, Thal DR, Trojanowski JQ, Vinters HV, Hyman BT, National Institute on Aging-Alzheimer’s Association. National Institute on Aging-Alzheimer’s Association guidelines for the neuropathologic assessment of Alzheimer’s disease: a practical approach. Acta Neuropathol. 2012;123:1–11. doi:10.1007/s00401-011-0910-3.
- Thal DR, Rüb U, Orantes M, Braak H. Phases of A beta-deposition in the human brain and its relevance for the development of AD. Neurology. 2002;58:1791–800.View ArticlePubMedGoogle Scholar
- Horie M, Watanabe K, Bepari AK, Nashimoto J, Araki K, Sano H, Chiken S, Nambu A, Ono K, Ikenaka K, Kakita A, Yamamura K, Takebayashi H. Disruption of actin-binding domain-containing Dystonin protein causes dystonia musculorum in mice. Eur J Neurosci. 2014;40:3458–71. doi:10.1111/ejn.12711.View ArticlePubMedGoogle Scholar
- Hasegawa M, Arai T, Nonaka T, Kametani F, Yoshida M, Hashizume Y, Beach TG, Buratti E, Baralle F, Morita M, Nakano I, Oda T, Tsuchiya K, Akiyama H. Phosphorylated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Ann Neurol. 2008;64:60–70. doi:10.1002/ana.21425.View ArticlePubMedPubMed CentralGoogle Scholar
- Kosaka T, Fu YJ, Shiga A, Ishidaira H, Tan CF, Tani T, Koike R, Onodera O, Nishizawa M, Kakita A, Takahashi H. Primary lateral sclerosis: upper-motor-predominant amyotrophic lateral sclerosis with frontotemporal lobar degeneration--immunohistochemical and biochemical analyses of TDP-43. Neuropathology. 2012;32:373–84. doi:10.1111/j.1440-1789.2011.01271.x.View ArticlePubMedGoogle Scholar
- Tsuji H, Arai T, Kametani F, Nonaka T, Yamashita M, Suzukake M, Hosokawa M, Yoshida M, Hatsuta H, Takao M, Saito Y, Murayama S, Akiyama H, Hasegawa M, Mann DM, Tamaoka A. Molecular analysis and biochemical classification of TDP-43 proteinopathy. Brain. 2012;135:3380–91. doi:10.1093/brain/aws230.View ArticlePubMedGoogle Scholar
- Takeuchi R, Toyoshima Y, Tada M, Tanaka H, Shimizu H, Shiga A, Miura T, Aoki K, Aikawa A, Ishizawa S, Ikeuchi T, Nishizawa M, Kakita A, Takahashi H. Globular Glial Mixed Four Repeat Tau and TDP-43 Proteinopathy with Motor Neuron Disease and Frontotemporal Dementia. Brain Pathol. 2016;26:82–94. doi:10.1111/bpa.12262.View ArticlePubMedGoogle Scholar
- Yokoseki A, Shiga A, Tan CF, Tagawa A, Kaneko H, Koyama A, Eguchi H, Tsujino A, Ikeuchi T, Kakita A, Okamoto K, Nishizawa M, Takahashi H, Onodera O. TDP-43 mutation in familial amyotrophic lateral sclerosis. Ann Neurol. 2008;63:538–42. doi:10.1002/ana.21392.View ArticlePubMedGoogle Scholar
- DeJesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M, Rutherford NJ, Nicholson AM, Finch NA, Flynn H, Adamson J, Kouri N, Wojtas A, Sengdy P, Hsiung GY, Karydas A, Seeley WW, Josephs KA, Coppola G, Geschwind DH, Wszolek ZK, Feldman H, Knopman DS, Petersen RC, Miller BL, Dickson DW, Boylan KB, Graff-Radford NR, Rademakers R. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron. 2011;72:245–56. doi:10.1016/j.neuron.2011.09.011.View ArticlePubMedPubMed CentralGoogle Scholar
- Konno T, Shiga A, Tsujino A, Sugai A, Kato T, Kanai K, Yokoseki A, Eguchi H, Kuwabara S, Nishizawa M, Takahashi H, Onodera O. Japanese amyotrophic lateral sclerosis patients with GGGGCC hexanucleotide repeat expansion in C9ORF72. J Neurol Neurosurg Psychiatry. 2013;84:398–401. doi:10.1136/jnnp-2012-302272.View ArticlePubMedGoogle Scholar
- Goldstein LH, Abrahams S. Changes in cognition and behavior in amyotrophic lateral sclerosis: nature of impairment and implications for assessment. Lancet Neurol. 2013;12:368–80. doi:10.1016/S1474-4422(13)70026-7.View ArticlePubMedGoogle Scholar
- Josephs KA, Stroh A, Dugger B, Dickson DW. Evaluation of subcortical pathology and clinical correlations in FTLD-U subtypes. Acta Neuropathol. 2009;118:349–58. doi:10.1007/s00401-009-0547-7.View ArticlePubMedPubMed CentralGoogle Scholar
- Tan RH, Kril JJ, Fatima M, McGeachie A, McCann H, Shepherd C, Forrest SL, Affleck A, Kwok JB, Hodges JR, Kiernan MC, Halliday GM. TDP-43 proteinopathies: pathological identification of brain regions differentiating clinical phenotypes. Brain. 2015;138:3110–22. doi:10.1093/brain/awv220.View ArticlePubMedGoogle Scholar
- Snowden J, Neary D, Mann D. Frontotemporal lobar degeneration: clinical and pathological relationships. Acta Neuropathol. 2007;114:31–8. doi:10.1007/s00401-007-0236-3.View ArticlePubMedGoogle Scholar
- Strong MJ, Grace GM, Freedman M, Lomen-Hoerth C, Woolley S, Goldstein LH, Murphy J, Shoesmith C, Rosenfeld J, Leigh PN, Bruijn L, Ince P, Figlewicz D. Consensus criteria for the diagnosis of frontotemporal cognitive and behavioural syndromes in amyotrophic lateral sclerosis. Amyotroph Lateral Scler. 2009;10:131–46. doi:10.1080/17482960802654364.View ArticlePubMedGoogle Scholar
- Liu-Yesucevitz L, Lin AY, Ebata A, Boon JY, Reid W, Xu YF, Kobrin K, Murphy GJ, Petrucelli L, Wolozin B. ALS-linked mutations enlarge TDP-43-enriched neuronal RNA granules in the dendritic arbor. J Neurosci. 2014;34:4167–74. doi:10.1523/JNEUROSCI.2350-13.2014.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang IF, Wu LS, Chang HY, Shen CK. TDP-43, the signature protein of FTLD-U, is a neuronal activity-responsive factor. J Neurochem. 2008;105:797–806. doi:10.1111/j.1471-4159.2007.05190.x.View ArticlePubMedGoogle Scholar
- Brettschneider J, Del Tredici K, Toledo JB, Robinson JL, Irwin DJ, Grossman M, Suh E, Van Deerlin VM, Wood EM, Baek Y, Kwong L, Lee EB, Elman L, McCluskey L, Fang L, Feldengut S, Ludolph AC, Lee VM, Braak H, Trojanowski JQ. Stages of pTDP-43 pathology in amyotrophic lateral sclerosis. Ann Neurol. 2013;74:20–38. doi:10.1002/ana.23937.View ArticlePubMedPubMed CentralGoogle Scholar
- Brettschneider J, Arai K, Del Tredici K, Toledo JB, Robinson JL, Lee EB, Kuwabara S, Shibuya K, Irwin DJ, Fang L, Van Deerlin VM, Elman L, McCluskey L, Ludolph AC, Lee VM, Braak H, Trojanowski JQ. TDP-43 pathology and neuronal loss in amyotrophic lateral sclerosis spinal cord. Acta Neuropathol. 2014;128:423–37. doi:10.1007/s00401-01299-6.View ArticlePubMedPubMed CentralGoogle Scholar
- Nishihira Y, Tan CF, Hoshi Y, Iwanaga K, Yamada M, Kawachi I, Tsujihata M, Hozumi I, Morita T, Onodera O, Nishizawa M, Kakita A, Takahashi H. Sporadic amyotrophic lateral sclerosis of long duration is associated with relatively mild TDP-43 pathology. Acta Neuropathol. 2009;117:45–53. doi:10.1007/s00401-008-0443-6.View ArticlePubMedGoogle Scholar
- Majounie E, Renton AE, Mok K, Dopper EG, Waite A, Rollinson S, Chiò A, Restagno G, Nicolaou N, Simon-Sanchez J, Van Swieten JC, Abramzon Y, Johnson JO, Sendtner M, Pamphlett R, Orrell RW, Mead S, Sidle KC, Houlden H, Rohrer JD, Morrison KE, Pall H, Talbot K, Ansorge O; Chromosome 9-ALS/FTD Consortium; French research network on FTLD/FTLD/ALS; ITALSGEN Consortium, Hernandez DG, Arepalli S, Sabatelli M, Mora G, Corbo M, Giannini F, Calvo A, Englund E, Borghero G, Floris GL, Remes AM, Laaksovirta H, McCluskey L, Trojanowski JQ, Van Deerlin VM, Schellenberg GD, Nalls MA, Drory VE, Lu CS, Yeh TH, Ishiura H, Takahashi Y, Tsuji S, Le Ber I, Brice A, Drepper C, Williams N, Kirby J, Shaw P, Hardy J, Tienari PJ, Heutink P, Morris HR, Pickering-Brown S, Traynor BJ. Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study. Lancet Neurol. 2012;11:323–30. doi:10.1016/S1474-4422(12)70043-1.View ArticlePubMedPubMed CentralGoogle Scholar
- Miyamoto R, Kawarai T, Oki R, Matsumoto S, Izumi Y, Kaji R. Lack of C9orf72 expansion in 406 sporadic and familial cases of idiopathic dystonia in Japan. Mov Disord. 2015;30:1430–1. doi:10.1002/mds.26310.View ArticlePubMedGoogle Scholar
- Ogaki K, Li Y, Atsuta N, Tomiyama H, Funayama M, Watanabe H, Nakamura R, Yoshino H, Yato S, Tamura A, Naito Y, Taniguchi A, Fujita K, Izumi Y, Kaji R, Hattori N, Sobue G. Analysis of C9orf72 repeat expansion in 563 Japanese patients with amyotrophic lateral sclerosis. Neurobiol Aging. 2012;33:2527.e11–16. doi:10.1016/j.neurobiolaging.2012.05.011.View ArticleGoogle Scholar
- Doi Y, Atsuta N, Sobue G, Morita M, Nakano I. Prevalence and incidence of amyotrophic lateral sclerosis in Japan. J Epidemiol. 2014;24:494–9.View ArticlePubMedGoogle Scholar
- Cronin S, Hardiman O, Traynor BJ. Ethnic variation in the incidence of ALS: a systematic review. Neurology. 2007;68:1002–7. doi:10.1212/01.wnl.0000258551.96893.6f.View ArticlePubMedGoogle Scholar