A harmonized classification system for FTLD-TDP pathology

Enrichment of C-Terminal Fragments in TAR DNA-Binding Protein-43 Cytoplasmic Inclusions in Brain but not in Spinal Cord of Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis

TDP-43 and FUS normal cytoplasmic functions are thought to involve regulated aggregation and disaggregation [1, 5, 8], similar to that of prion proteins, where aggregation occurs by a self-templating process, likely involving properties of amyloid, or beta pleated sheet structure. Both thioflavin-T and thioflavin-S fluoresce when bound to amyloid fibrils [7]. We previously showed that TDP-43 peptides form amyloidogenic fibrils, binding to thioflavin-T [3]. Recently, TDP-43-positive lower motor neuron (LMN) inclusions in 28% of 47 cases of ALS, but no inclusions in 22 FTLD-TDP cases, were shown to be positive with thioflavin-S [6]. Using thioflavin-S, we surveyed brain tissues from 44 cases, including FTLD-TDP type A, (17 cases), type B (14 cases), type C (3 cases), sporadic ALS (2 cases) familial ALS (FALS) (2 each with SOD1 and C9orf72 mutations), and FTLD-FUS, including atypical FTLD-U (aFTLD-U, 2 cases) and basophilic inclusion body disease (BIBD, 2 cases). Our routine modified thioflavin-S stain includes pretreatment of tissue sections with potassium permanganate and bleaching with potassium metabisulfite and oxalic acid followed by treatment with sodium hydroxide and hydrogen peroxide, removing lipid autofluorescence and resulting in improved definition of pathological lesions. Slides were viewed using a Nikon BV-2A filter cube. Confocal microscopy was performed using thioflavin-S staining as above and phospho TDP43 (pS409/410-1). Inclusions in most cases of FTLD-TDP and ALS were thioflavin-S positive. The density and distribution of thioflavin-S positive inclusions was similar to that seen with ubiquitin and fewer than with TDP-43 immunohistochemistry. In FTLD-TDP type A, neuronal intranuclear inclusions (NIIs), neuronal cytoplasmic inclusions (NCIs) and dystrophic neurites (DNs) were strongly fluorescent (Fig. 1a). Fluorescent confocal microscopy showed co-localization of thioflavin-S with TDP-43 positive inclusions (Fig. 1b). In FTLD-TDP type B, rare NCIs in all layers of cortex were positive but dentate gyrus inclusions were easily seen (Fig. 1c). In FTLD-TDP type C, long DNs in cortex were strongly thioflavin-S positive (Fig. 1d). Inclusions in FTLD-FUS were negative with thioflavin-S; while rare hippocampal dentate gyrus neurons had fluorescent granular inclusions, large cytoplasmic inclusions and intranuclear vermiform inclusions were not seen with thioflavin-S (Fig. 2). Skein-like inclusions (SLI) and Lewy-like bodies (LLB) in LMNs of ALS cases were positive with thioflavin-S (Fig. 3). The two cases of FALS with SOD1 mutations were not thioflavin-S positive. Additionally, eight of the 17 cases of FTLD-TDP type A had exuberant thioflavin-S positive astrocytosis, making identification of TDP-43 pathology problematic in five of these (Fig. 4). Fig. 1 Superficial frontal cortex, FTLD-TDP type A (1a). Numerous thioflavin-S positive c-shaped and annular NCIs and a few DNs. Inset: thioflavin-S positive NII. Thioflavin-S, 400x. Confocal microscopy (1b). Same case as Fig. 1a, with thioflavin-S (green, left), ... Fig. 2 Motor cortex, FTLD-FUS (BIBD) (2a). Large cytoplasmic upper motor neuron inclusion on left, consistent with basophilic inclusion body (BIB), negative with thioflavin-S. BIB on right immunolabeled with FUS for comparison. Thioflavin-S stain and FUS immunohistochemistry, ... Fig. 3 Lumbar anterior horn, ALS. Lower motor neurons with LLB (left) and SLIs (middle and right). Scattered shrunken neurons nearby (middle and right images) contain compressed SLIs. Thioflavin-S, 600x. Fig. 4 Superficial frontal cortex, FTLD-TDP type A. Exuberant thioflavin-S positive astrocytosis. Thioflavin-S, 200x. There are at least three possible reasons that these results differ from those of Robinson et al. The modified thioflavin-S protocol used in the current study provides results that are superior to other thioflavin-S protocols, the BV-2A filter produces the brightest images, and the brain tissue is briefly paraformaldehyde-fixed (30 hours). Fixation does not likely impact results, but we cannot exclude that possibility. Clearly more cases need to be analyzed with thioflavin-S, and that work is in progress. Nonetheless, it is important that thus far, inclusions in most cases of FTLD-TDP, but not FTLD-FUS or SOD1 FALS, are thioflavin-S positive, suggesting that the prion-like property of TDP-43 may be involved in FTLD-TDP and sporadic ALS and non-SOD1 FALS, but that this may not be the case in FTLD-FUS. The significance of the thioflavin-S positive astrocytosis in some cases of FTLD-TDP type A is currently under investigation. In addition, the fact that TDP-43 in FTLD-TDP has properties of amyloid has implications for the interpretation of amyloid imaging studies [2], as Pittsburgh Compound-B is a modified thioflavin-T derivative [4].

Perry syndrome is a rare atypical parkinsonism with depression, apathy, weight loss, and central hypoventilation caused by mutations in dynactin p150glued (DCTN1). A rare distal hereditary motor neuropathy, HMN7B, also has mutations in DCTN1. Perry syndrome has TAR DNA-binding protein of 43 kDa (TDP-43) inclusions as a defining feature. Other TDP-43 proteinopathies include amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with and without motor neuron disease (FTLD-MND). TDP-43 forms aggregates in neuronal cytoplasmic inclusions (NCIs), neuronal intranuclear inclusions, dystrophic neurites (DNs), as well as axonal spheroids, oligodendroglial cytoplasmic inclusions, and perivascular astrocytic inclusions (PVIs). We performed semiquantitative assessment of these lesions and presence of dynactin subunit p50 lesions in 3 cases of Perry syndrome and one of HMN7B. We compared them with 3 cases of FTLD-MND, 3 of ALS, and 3 of hippocampal sclerosis (HpScl). Perry syndrome had NCIs, DNs, and frequent PVIs and spheroids. Perry syndrome cases were similar, but different from ALS, FTLD-MND, and HpScl. TDP-43 pathology was not detected in HMN7B. Dynactin p50 inclusions were observed in both Perry syndrome and HMN7B, but not in the other conditions. These results suggest that Perry syndrome may be distinctive type of TDP-43 proteinopathy.

Valosin-containing protein (VCP) is involved in multiple cellular activities. Mutations in VCP lead to heterogeneous clinical presentations including inclusion body myopathy with Paget's disease of the bone, frontotemporal dementia and amyotrophic lateral sclerosis, even in patients carrying the same mutation. We screened a cohort of 48 patients with familial frontotemporal dementia (FTD) negative for MAPT, GRN, and C9orf72 mutations for other known FTD genes by using whole exome sequencing. In addition, we carried out targeted sequencing of a cohort of 37 patients with frontotemporal lobar degeneration with Transactive response DNA-binding protein 43 (TDP-43) subtype from the Netherlands Brain bank. Two novel (p.Thr262Ser and p.Arg159Ser) and one reported (p.Met158Val) VCP mutations in three patients with a clinical diagnosis of FTD were identified, and were absence in population-match controls. All three patients presented with behavioral changes, with additional semantic deficits in one. No signs of Paget or muscle disease were observed. Pathological examination of the patient with VCP p.Arg159Ser mutation showed numerous TDP-43 immunoreactive (IR) neuronal intranuclear inclusions (NII) and dystrophic neurites (DN), while a lower number of NII and DN were observed in the patient with the VCP p.Thr262Ser mutation. Pathological findings of both patients were consistent with FTLD-TDP subtype D. Furthermore, only rare VCP-IR NII was observed in both cases. Our study expands the clinical heterogeneity of VCP mutations carriers, and indicates that other additional factors, such as genetic modifiers, may determine the clinical phenotype.

Frontotemporal lobar degeneration (FTLD) encompasses a heterogeneous group of clinical syndromes that include frontotemporal dementia (FTD), frontotemporal dementia with motor neurone disease (FTD/MND), progressive non-fluent aphasia (PNFA), semantic dementia (SD) and progressive apraxia (PAX). Clinical phenotype is often assumed to be a poor predictor of underlying histopathology. Advances in immunohistochemistry provide the opportunity to re-examine this assumption. We classified pathological material from 79 FTLD brains, blind to clinical diagnosis, according to topography of brain atrophy and immunohistochemical characteristics. There were highly significant relationships to clinical syndrome. Atrophy was predominantly frontal and anterior temporal in FTD, frontal in FTD/MND, markedly asymmetric perisylvian in PNFA, asymmetric bitemporal in SD and premotor, parietal in PAX. Tau pathology was found in half of FTD and all PAX cases but in no FTD/MND or SD cases and only rarely in PNFA. FTD/MND, SD and PNFA cases were ubiquitin and TDP-43 positive. SD cases were associated with dystrophic neurites without neuronal cytoplasmic or intranuclear inclusions (FTLD-U, type 1), FTD/MND with numerous neuronal cytoplasmic inclusions (FTLD-U, type 2 ) and PNFA with neuronal cytoplasmic inclusions, dystrophic neurites and neuronal intranuclear inclusions (FTLD-U, type 3). MAPT mutations were linked to FTD and PGRN mutations to FTD and PNFA. The findings demonstrate predictable relationships between clinical phenotype and both topographical distribution of brain atrophy and immunohistochemical characteristics. The findings emphasise the importance of refined delineation of both clinical and pathological phenotype in furthering understanding of FTLD and its molecular substrate.

Frontotemporal lobar degeneration (FTLD) without tau pathology is clinically and pathologically heterogeneous. The present report describes the neuropathology of 52 brains with FTLD without tau pathology compared with 10 brains of amyotrophic lateral sclerosis (ALS) without dementia using ubiquitin immunohistochemistry. The 52 cases were classified into 47 cases of FTLD with motor neuron disease (MND)-type inclusions but without MND (FTLD-MNI), three cases of FTLD with MND (FTLD-MND), and two cases of dementia lacking distinctive histopathology (DLDH) based on the features of ubiquitin-immunoreactive (ubiquitin-ir) structures in the caudate, frontotemporal cortices and dentate fascia, and presence or absence of neuronal loss in lower motor neurons. Many ubiquitin-ir neuronal inclusions and neurites in the caudate nucleus, frontotemporal cortices, and ubiquitin-ir crescent-or ring-shaped neuronal inclusions in the dentate fascia characterized FTLD-MNI. Ubiquitin-ir neuronal intranuclear inclusions (NII) were observed in 26 of 43 cases and associated with many neurites in the caudate nucleus as well as a familial history in most cases. A subset of cases had Pick-body-like inclusions in the dentate fascia and caudate nucleus with paucity of neuritic pathology and no NII; another had crescent-shaped inclusions in the dentate fascia and neuritic pathology with NII in the caudate. FTLD with MND was characterized by a few or no ubiquitin-ir inclusions in the caudate nucleus and frontotemporal cortices and ubiquitin-ir granular inclusions in the dentate fascia, as well as loss of lower motor neurons. These features were similar to ALS, but different from FTLD-MNI. The findings suggest that FTLD-MNI has a different pathogenesis from FTLD-MND and ALS.

Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are 2 neurodegenerative disorders that share clinical, genetic, and neuropathologic features. The presence of abnormal expansions of GGGGCC repeats (G4C2 repeats) in a noncoding region of the Chromosome 9 open reading frame 72 (C9orf72) gene is the major genetic cause of both FTLD and ALS. Transcribed G4C2 repeats can form nuclear RNA foci and recruit RNA-binding proteins, thereby inhibiting their normal function. Moreover, through a repeat-associated non-ATG translation mechanism, G4C2 repeats translation leads to dipeptide-repeat protein aggregation in the cytoplasm of neurons. Here, we identify Drosha protein as a new component of these dipeptide-repeat aggregates. In C9orf72 mutation cases of FTLD-TDP (c9FTLD-TDP) and ALS (c9ALS), but not in FTLD or ALS cases without C9orf72 mutation, Drosha is mislocalized to form neuronal cytoplasmic inclusions in the hippocampus, frontal cortex, and cerebellum. Further characterization of Drosha-positive neuronal cytoplasmic inclusions in the hippocampus, frontal cortex, and cerebellum revealed colocalization with p62 and ubiquilin-2, 2 pathognomonic signatures of c9FTLD-TDP and c9ALS cases; however, Drosha inclusions rarely colocalized with TDP-43 pathology. We conclude that Drosha may play a unique pathogenic role in the onset or progression of FTLD-TDP/ALS in patients with the C9orf72 mutation.

Pathological 43-kDa transactive responsive DNA-binding protein (TDP-43) has been identified as the major, i.e. disease defining, pathological protein in the majority of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) or frontotemporal lobar degeneration (FTLD) with ubiquitin-positive inclusions (FTLD-U) with or without motor neuron disease (MND), which is known now as FTLD-TDP (1,2). ALS has been linked to FTLD through TDP-43 pathology that may be widespread in the central nervous system (CNS), thereby creating a new concept of TDP-43 proteinopathy as a disease continuum that encompasses ALS and FTLD as well as rarer conditions such as progressive muscular atrophy or primary lateral sclerosis (3, for review see (4)). TDP-43 pathology in addition, or ‘secondary’, to disease defining inclusion pathologies may occur in other neurodegenerative diseases such as tauopathies or α-synulceinopathies (4). A subgroup of ALS and FTLD cases with neuronal inclusions composed of an ubiquitinated protein, negative for pathological TDP-43, tau, or α-synuclein, was shown to be characterized by the pathological aggregation of a protein called fused in sarcoma (FUS), which shows functional homology to TDP-43 (5–7). Previous to the identification of these two disease defining proteins, there have been reports of single cases of sporadic or familiar ALS or FTLD cases showing pathological tau aggregations (or neurofibrillary tangles) as their pathological hallmark feature with (8) or without (9–14) the presence of additional ubiquitinated inclusions, published by research groups in continental Europe and Asia. The tau pathology in these cases is in part reminiscent of the Parkinsonism-dementia complex and ALS (ALS/PDC) occurring in geographic isolates such as the Kii Peninsula of Japan (15) or Guam in the Mariana Islands (16–18). These are tauopathies that differ from Alzheimer’s disease (AD) in terms of their topographical distribution of tau pathology and the lower level of amyloid-β depositions (19). We here report a patient with sporadic ALS and eye closure apraxia from the continental United States showing multisystem tau pathology atypical for AD by morphology in the brain and spinal cord. There also were robust cortical amyloid-β deposits, but no pathognomonic TDP-43, FUS or α-synuclein pathology. We conclude that tau pathology alone is sufficient to cause ALS and FLTD-MND-like neurodegeneration in the continental United States suggesting that, in part, similar pathogenetic mechanisms might be involved in the patient described in this report, as in ALS occurring in the Kii Peninsula and Guam.

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0335-2) contains supplementary material, which is available to authorized users.

FIG4 is a phosphatase that regulates intracellular vesicle trafficking along the endosomal-lysosomal pathway. Mutations of FIG4 lead to the development of Charcot-Marie-Tooth disease type 4J and amyotrophic lateral sclerosis (ALS). Moreover, ALS-associated proteins (transactivation response DNA protein 43 (TDP-43), fused in sarcoma (FUS), optineurin, ubiquilin-2, charged mutivesicular body protein 2b (CHMP2B) and valosin-containing protein) are involved in inclusion body formation in several neurodegenerative diseases. Using immunohistochemistry, we examined the brains and spinal cords of patients with various neurodegenerative diseases, including sporadic TDP-43 proteinopathy (ALS and frontotemporal lobar degeneration). TDP-43 proteinopathy demonstrated no FIG4 immunoreactivity in neuronal inclusions. However, FIG4 immunoreactivity was present in Pick bodies in Pick's disease, Lewy bodies in Parkinson's disease and dementia with Lewy bodies, neuronal nuclear inclusions in polyglutamine and intranuclear inclusion body diseases, and Marinesco and Hirano bodies in aged control subjects. These findings suggest that FIG4 is not incorporated in TDP-43 inclusions and that it may have a common role in the formation or degradation of neuronal cytoplasmic and nuclear inclusions in several neurodegenerative diseases.

Research on familial frontotemporal lobar degeneration (FTLD) has led to the discovery of disease-causing genes: microtubule-associated protein tau (MAPT), progranulin (PGRN) and valosin-containing protein (VCP). TAR DNA-binding protein of 43 kDa (TDP-43) has been identified as a major component of tau-negative ubiquitin-positive inclusions in familial and sporadic FTLD and amyotrophic lateral sclerosis (ALS), which are now referred to as TDP-43 proteinopathy. Recent findings of mutations in TDP-43 gene in familial and sporadic ALS cases confirm the pathogenetic role for TDP-43 in neurodegeneration. TDP-43 proteinopathies have been classified into 4 pathological subtypes. Type 1 is characterized by numerous dystrophic neurites (DNs), Type 2 has numerous neuronal cytoplasmic inclusions (NCIs), Type 3 has NCIs and DNs and Type 4 has neuronal intranuclear inclusions (NIIs) and DNs. There is a close relationship between such pathological subtypes of TDP-43 proteinopathy and the immunoblot pattern of C-terminal fragments of accumulated TDP-43. These results parallel our earlier findings of differing C-terminal tau fragments in progressive supranuclear palsy and corticobasal degeneration, despite identical composition of tau isoforms. Taken together, these results suggest that elucidating the mechanism of C-terminal fragment origination may shed light on the pathogenesis of several neurodegenerative disorders involving TDP-43 proteinopathy and tauopathy.

TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis

Psychiatric symptoms and TDP-43 pathology in amyotrophic lateral sclerosis

Neuronal intermediate filament inclusion disease (NIFID), a rare form of frontotemporal lobar degeneration (FTLD), is characterized neuropathologically by focal atrophy of the frontal and temporal lobes, neuronal loss, gliosis, and neuronal cytoplasmic inclusions (NCI) containing epitopes of ubiquitin and neuronal intermediate filament proteins. Recently, the 'fused in sarcoma' (FUS) protein (encoded by the FUS gene) has been shown to be a component of the inclusions of familial amyotrophic lateral sclerosis with FUS mutation, NIFID, basophilic inclusion body disease, and atypical FTLD with ubiquitin-immunoreactive inclusions (aFTLD-U). To further characterize FUS proteinopathy in NIFID, and to determine whether the pathology revealed by FUS immunohistochemistry (IHC) is more extensive than α-internexin, we have undertaken a quantitative assessment of ten clinically and neuropathologically well-characterized cases using FUS IHC. The densities of NCI were greatest in the dentate gyrus (DG) and in sectors CA1/2 of the hippocampus. Anti-FUS antibodies also labeled glial inclusions (GI), neuronal intranuclear inclusions (NII), and dystrophic neurites (DN). Vacuolation was extensive across upper and lower cortical layers. Significantly greater densities of abnormally enlarged neurons and glial cell nuclei were present in the lower compared with the upper cortical laminae. FUS IHC revealed significantly greater numbers of NCI in all brain regions especially the DG. Our data suggest: (1) significant densities of FUS-immunoreactive NCI in NIFID especially in the DG and CA1/2; (2) infrequent FUS-immunoreactive GI, NII, and DN; (3) widely distributed vacuolation across the cortex, and (4) significantly more NCI revealed by FUS than α-internexin IHC.

Machado-Joseph disease (MJD) is a dominantly inherited spinocerebellar disorder, and expansions of trinucleotide (CAG) at chromosome 14 have been shown to be the locus of this disorder. Polyglutamine CAG stretches in the neuronal cytoplasms and nuclei were studied with immunolabeling using 1C2, a monoclonal antibody recognizing polyglutamine stretches, and polyclonal antiubiquitin antibody in six genetically verified cases of MJD. 1C2 clearly labeled two types of neuronal intranuclear inclusions (NII) and neuronal cytoplasmic inclusions (NCI) in the substantia nigra, pontine nucleus, dentate nucleus and spinal anterior horn where NII and NCI were also positive for ubiquitin, as were extracellular dot-like structures and oligodendroglial inclusions. 1C2-positive NII and NCI had a lesion-specific distribution. While the spinal motoneurons contained only 1C2-positive NCI and lacked 1C2-positive NII, the ventral pontine nucleus neurons had many 1C2-positive NII and few 1C2-positive NCI. Semi-quantitative examination of NII and NCI positive for 1C2 or ubiquitin demonstrated that there were more 1C2-positive NII and NCI than ubiquitin-positive ones. It is noteworthy that the nuclei of the spinal motoneurons lacked 1C2-positive immunoreactivity, so that ubiquitination of 1C2-positive structures is presumed to occur late in the course of the disease.