Invited review: Neuropathology of tauopathies: principles and practice

Neuropathology and Applied Neurobiology (2015), 41, 3 23 doi: 10.1111/nan.12208 Invited review: Neuropathology of tauopathies: principles and practice G. G. Kovacs Institute of Neurology, Medical University of Vienna, Vienna, Austria G. G. Kovacs (2015) Neuropathology and Applied Neurobiology 41, 3 23 Neuropathology of tauopathies: principles and practice Tauopathies are clinically, morphologically and biochemically heterogeneous neurodegenerative diseases characterized by the deposition of abnormal tau protein in the brain. The neuropathological phenotypes are distinguished based on the involvement of different anatomical areas, cell types and presence of distinct isoforms of tau in the pathological deposits. The nomenclature of primary tauopathies overlaps with the modern classification of frontotemporal lobar degeneration. Neuropathological phenotypes comprise Pick s disease, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, primary age-related tauopathy, formerly called also as neurofibrillary tangleonly dementia, and a recently characterized entity called globular glial tauopathy. Mutations in the gene encoding the microtubule-associated protein tau are associated with frontotemporal dementia and parkinsonism linked to chromosome 17. In addition, further neurodegenerative conditions with diverse aetiologies may be associated with tau pathologies. Thus, the spectrum of tau pathologies and tauopathy entities expands beyond the traditionally discussed disease forms. Detailed multidisciplinary studies are still required to understand their significance. Keywords: argyrophilic grain disease, corticobasal degeneration, globular glial tauopathy, Pick s disease, primary agerelated tauopathy, progressive supranuclear palsy Introduction and scope of review Correspondence: Gabor G. Kovacs, Institute of Neurology, Medical University of Vienna, AKH 4J, Währinger Gürtel 18-20, A-1097 Vienna, Austria. Tel: +43 1 40400 55070; Fax +43 1 40400 55110; E-mail: gabor.kovacs@meduniwien.ac.at [Correction added on 09 February 2015, after online publication: Some corrections were omitted during production and this has now been fixed in both the Print and Online version of this article.] Tauopathies are clinically, morphologically and biochemically heterogeneous neurodegenerative diseases characterized by the deposition of abnormal tau protein in the brain. The neuropathological phenotypes are distinguished based on the involvement of different anatomical areas, cell types and presence of distinct isoforms of tau in the pathological deposits. The nomenclature of classical phenotypes associated with primary tauopathies overlaps with the modern classification of frontotemporal lobar degeneration [1]. Neuropathological phenotypes comprise Pick s disease (PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic grain disease (AGD), primary age-related tauopathy (PART), formerly known as neurofibrillary tangle-only dementia (NFT-dementia) and a recently characterized entity called globular glial tauopathy (GGT). Mutations in the encoding gene of the microtubule-associated protein tau (MAPT) are associated with frontotemporal dementia and parkinsonism linked to chromosome 17. In addition, several neurodegenerative conditions with diverse aetiologies may be associated with tau pathology; some of these are known as secondary tauopathies, as other proteins play a central role in their pathogenesis. Alzheimer s disease (AD) is one major neurodegenerative disorder showing neuronal tau pathology; however, as it is characterized by further pathologies, in particular the deposition of amyloid-β (Aβ), it is not discussed in detail here. The aim of the present review is to provide an overview of the 3

4 G. G. Kovacs current concepts of the neuropathology of tauopathies with a focus on diagnostic aspects. Fundamentals of tauopathies Tau is a MAP binding to microtubules and promoting their polymerization [2]. It plays an important role in maintaining axonal transport and neuronal integrity but has a physiological role in dendrites, and it is expressed at low levels in glial cells. Non-microtubular localizations for tau have also been reported [3]. In the adult human brain, six isoforms of tau are expressed by alternative splicing from the MAPT gene located on chromosome 17q21 [4,5]. Mutations in the MAPT gene lead to hereditary diseases associated with the accumulation of pathological tau protein; more than 40 mutations have been described [6]. Tau isoforms, ranging from 352 to 441 amino acids, are generated by alternative splicing of exons 2, 3 and 10. The six isoforms differ from each other by the presence or absence of 29- or 58-amino acid inserts in the N-terminal part and by the presence of either three (3R) or four (4R) tandem repeat sequences of 31 or 32 amino acids. The repeat regions are binding domains that mediate the interaction between tau and microtubules. In the normal human brain, similar levels of 3R and 4R isoforms are expressed. Tau filaments in patients affected by tauopathies are composed of either 3R- or 4R-tau or both, reflecting the biochemical heterogeneity of tauopathies [3,7]; furthermore, tau composition provides the basis of the current molecular classification of tauopathies (Figure 1). The most important posttranslational modification of tau in disease is hyperphosphorylation, which has serious functional effects [8]. Normal tau is phosphorylated on two or three residues in contrast to hyperphosphorylated tau that is phosphorylated at least on eight residues, but there are further epitopes that may theoretically be phosphorylated [9]. Phosphorylation of tau has an impact on microtubule stability and axonal transport, dendritic positioning and synaptic health, cell signalling at plasma membranes, protection of DNA from cells stressors and release of tau [9]. Further posttranslational modifications of tau comprise N- and C-terminal truncation, acetylation, glycosylation, oxidative and nitrative injuries, transglutamination, deamidation and formation of tau oligomers [10]. Although most of the morphological and biochemical studies focus on a few phosphorylation Figure 1. Molecular classification of tauopathies (FTLD-Tau) and major patterns of insoluble tau observed on Western blotting. PiD, Pick s disease; PSP, progressive supranuclear palsy; CBD, corticobasal degeneration; AGD, argyrophilic grain disease; GGT, globular glial tauopathy; PART, primary age-related tauopathy. epitopes and isoforms of tau, our knowledge expands and further modifications may prove to be important for neuropathological and in vivo biomarker diagnostic practice. Indeed, it has recently been reported that tau acetylation is present in tau pathologies related to AD and several tauopathies, including PiD, GGT, PSP, CBD, chronic traumatic encephalopathy (CTE) and unclassifiable 4R tauopathies, but not in AGD-related tau pathologies [11,12]. It was speculated that acetylation may represent a turning point that accelerates tau toxicity; thus, its lack could be a reason why AGD is most often associated with a non-progressive medial temporal lobe pathology [11]. Clinical symptoms related to sporadic tauopathies It is generally true for neurodegenerative diseases that different disorders can affect the same anatomical regions. During the progression of disease an increasing number of anatomical regions will be affected, which leads to complex constellations of symptoms. The clinical symptoms are determined by the system affected and do not unequivocally reflect the molecular pathologic background. Thus, not only tauopathies, but also other forms of neurodegenerative disorders associated with other conformationally altered proteins [e.g. α-synuclein;

Neuropathology of tauopathies 5 (TAR)DNA-binding protein 43 (TDP-43); fused in sarcoma; prion protein; Aβ] may show similar clinical symptoms. This means that when the neuropathologist reads the clinical information, several disorders may be anticipated. There are, however, certain constellations of symptoms that may be suggestive of a specific tauopathy. It is difficult to provide a typical age at onset of disorders, due also to the fact that many historical reports lack genetic analysis and differ in the definition of disease. For example, PiD, PSP, CBD and GGT were considered to be presenile disorders, however, recent studies described these in the elderly as well [13 18]. Most forms of tauopathy, in particular PiD, PSP, CBD and GGT, may be associated with clinical features of frontotemporal dementia (FTD) including progressive aphasia. FTD often associates with an extrapyramidal movement disorder or with motor neurone disease (MND). Secondary symptoms are observed during the course of the disease. The classical clinical presentation of PSP is referred to as Richardson or PSP-syndrome (i.e. Steele Richardson Olszewski syndrome) [19]. However, PSPtype pathology may be associated with the clinical corticobasal syndrome (CBS) or apraxia of speech. Furthermore, brainstem-predominant PSP [20] clinically presents as PSP-P (where P refers to parkinsonism), or pure akinesia with gait freezing [21]. Some patients may show progressive cerebellar ataxia. On the other hand, CBD type pathology, which mostly associates with CBS, may present with Richardson syndrome, posterior cortical atrophy syndrome, dementia with features similar to AD [22] or rarely cerebellar ataxia [23]. PiD neuropathological phenotype is seen in patients presenting most often with clinical phenotypes of FTD [17,24]. Rarely PiD type neuropathology is seen in individuals with an AD-like amnestic syndrome [17]. A few studies on the clinical aspects of AGD report progressive cognitive decline, urinary incontinence, memory disturbances and personality changes often with aggression and ill temper [25]. Rarely the prominent abnormal behaviour and aggression or aphasia may raise the suspicion of FTD [26,27]. Symptoms in persons with PART (NFT-dementia) usually range from normal to amnestic cognitive changes; in the latter group disorientation is frequent, and depression and paranoid ideas may also be observed [28]. Compared with the recently suggested limbic predominant form of AD [29], PART (NFT-dementia) patients usually have an older age at disease onset and shorter disease duration [30]. Although there are descriptions of the association of PSP (or CBS) with corticospinal tract degeneration or parkinsonism with MND [31,32], these syndromes are now included in the neuropathological group of GGTs [33]. Genetic aspects of tauopathies An important step for the full characterization of tauopathies is the genetic analysis of MAPT. Mutations in the MAPT may be associated with the neuropathological phenotypes of PiD, PSP and CBD, but also show distinctive morphologies. The extended haplotype (H1/H2) of the MAPT gene is of interest in sporadic tauopathies. Inheritance of the H1 haplotype and the H1/H1 genotype is a recognized risk factor for PSP [34]. The H1c subhaplotype confers additional risk for PSP. Either the H1 haplotype or the H1c subhaplotype also confers increased risk for CBD [22]. This haplotype is not associated with PiD [35]. Similarly to PSP and CBD, the frequency of MAPT H1/H1 genotype tends to be higher in AGD [36] and also in NFTdementia [30,37]. In GGT cases, the H1/H1 genotype was found in eight out of nine cases examined (one from Japan) and H1/H2 was found in a further case. Some studies report the evaluation of apolipoprotein E alleles (APOE). Regarding APOE ε4 carrier state: in AGD, it is lower than in AD; moreover, the incidence of ε2 allele is higher in AGD [25]. The APOE ε4 carrier state is low in NFT-dementia as compared with the limbic predominant form of AD [30,37]. In GGT, mainly ε3 alleles are observed [38]. Methods for the characterization of tauopathies Light microscopy methods For the neuropathological diagnostic practice detection of hyperphosphorylated tau and distinguishing tau isoforms are the most relevant. There are several well-characterized, commercially available antibodies, which can be used for immunohistochemistry. These include phosphorylationdependent antibodies; antibodies against truncated tau; or against conformational modifications. Importantly, phosphorylation sites may be distinct in different anatomical regions, and their temporal distribution may also differ in tauopathy entities [39,40]. This means that different phospho-tau antibodies can detect pathological structures distinctly in the same brain. This might have implications for diagnostic practice. The most widely used phospho-tau

6 G. G. Kovacs Table 1. A list of antibodies and stainings used for the evaluation of tauopathies Staining/antibody Remarks Primarily used in the diagnostic practice to characterize tau pathologies AT8 ph-ser202/thr205 AT100 ph-thr212/ser214: detects fibrillar tau RD4 4R-tau isoform (buffer ph 6.0 followed by 98% formic acid for 1 min as epitope retrieval) RD3 3R-tau isoform (buffer ph 6.0 followed by 98% formic acid for 1 min as epitope retrieval) p62 Clone 3/P62 LCK LIGAND (buffer ph 6.0 as epitope retrieval) Ubiquitin Clone Ubi-1 (buffer ph 6.0 as epitope retrieval) Gallyas silver Stains most of the pathological fibrillar tau+ve structures Bielschowsky silver Evaluation of neuritic plaques and axonal pathology Examples of tau antibodies available for specific characterization of tau pathologies AT180 ph-thr231 AT270 ph-thr181 Alz50 Misfolded conformation of tau (aa 2 10 and 312 342) CP3 ph-ser214 CP13 ph-ser202 12E8 ph-ser262/ser356 PG5 ph-ser409 PHF-1 ph-ser396/ser404 9G3 ph-tyr18 MC1 Conformation dependent antibody (aa 312 322) T46.1 Amino acids 428 441 (C-terminus) Tau-C3 Tau truncated at aspartic acid 421 MAb 359 Aacetylate-tau at lysine residue 274 ac-k280 Acetylated-tau at lysine 280 The epitope retrieval methods are provided according to the author s experience. Note that further polyclonal and monoclonal antibodies are available for research and diagnostics. antibody is AT8 (Ser202/Thr205); in the present review, when not otherwise specified, the immunostaining pattern seen with the AT8 antibody is referred to as tau immunoreactivity. Tau isoform-specific (RD3 and RD4) monoclonal antibodies are also available [41]. Additional stains useful for the characterization of tau pathologies include silver stains (Bielschowsky, Bodian, Gallyas, Campbell Switzer) [42], thioflavine-s staining and immunostaining for ubiquitin or p62. Gallyas silver staining together with AT100 immunohistochemistry (i.e. Thr212/Ser214) are important tools to investigate whether the pathological tau deposits are of filamentous nature or not [43]. Application of silver techniques has been hindered by the difficulties of the techniques and lack of standardized protocols [44]. In diagnostic practice ubiquitin and p62/sequestome-1 immunohistochemistry takes the place of these silver stainings [45]. It must be kept in mind, however, that the amount of tauimmunoreactivity using AT8 exceeds that seen with other staining methods (i.e. silver staining or p62/ubiquitin immunostaining). Antibodies and staining used in diagnostic practice and research are summarized in Table 1. Some of these antibodies may require specific pretreatments depending on the length of formalin fixation [46,47]. Electron microscopy Sarcosyl-insoluble tau dispersed filament preparations or Karnovsky (glutaraldehyde/paraformaldehyde) fixed brain samples can be examined ultrastructurally, including using immunogold electron microscopic examination [48]. The morphology of filaments in preparations appears as paired helical filaments, straight filaments, tubular profiles or twisted ribbon-like structures. Immunoblotting Approaches for the examination of frozen brain samples by immunoblotting comprise the evaluation of banding patterns of soluble and insoluble tau and dephosphorylated tau preparations [7,49]. Patterns of insoluble tau observed are (I) major bands at 60, 64 and 68 kda (e.g. in AD and NFT-dementia); (II) bands at 64 and

Neuropathology of tauopathies 7 68 kda (e.g. in 4R predominant tauopathies, AGD, PSP, CBD and GGT); and (III) bands at 60 and 64 kda (e.g. in 3R predominant tauopathies and PiD) (Figure 1). A minor band at 72 kda is usually associated with pattern I. Tauopathies associated with mutations in the MAPT gene may show any of the patterns and isoform predominance. It was suggested that different proteolytic processing of abnormal tau takes place in PSP and CBD, which is also detectable by immunoblotting [50]. Morphological features of tau pathologies Before starting the neuropathological evaluation, one must be familiar with the cellular distribution of immunoreactive structures visible using immunohistochemistry for phospho-tau (in particular the AT8 antibody) or isoform specific antibodies (Figure 2 and Table 2). Tau immunoreactivity related to neurones includes (i) pretangles, (ii) NFTs, (iii) Pick bodies, (iv) spherical cytoplasmic inclusions, (v) dystrophic neurites, (vi) threads and (vii) grains. These structures show variable silver staining or ubiquitin/p62 immunoreactivity. For example, pretangles are thought to represent an early stage of development into ubiquitinated NFTs [51]. A hierarchical model of plaque biogenesis was proposed, which suggests that plaque-associated dystrophic neurites develop in a particular sequence, thus tau-immunoreactivity of neuritic plaques varies between regions in the same brain [52 54]. It must be noted that small (2 5 μm), rounded, tau-ve neuritic profiles, which are usually not argyrophilic but can be seen in ubiquitin and p62 immunostaining, can be observed without amyloid plaques in non-diseased brains [55]. Astrocytes show a variety of tau immunoreactivity, often labelled with different terminologies. For diagnostic practice differentiation of tufted astrocytes (PSP) from astrocytic plaques (CBD) is the most relevant. Further tau immunoreactive entities (AT8) comprise thorn-shaped and the recently characterized globular astroglial inclusions (GAIs) [33,56]. In addition, diffuse fine granular tau immunopositivity along astrocytic processes is observed in elderly individuals in the temporal lobe. Similar profiles are often termed bush-like astrocytes in AGD. Because these are non-argyrophilic, they are likely to represent hyperphosphorylated tau, preceding aggregation into fibrillary tau [57 59]. The term ramified astrocyte, referring to cells with eccentric nuclei and Gallyas-positive branched thick processes, was used to describe taupositive astrocytes in PiD. Interestingly, a recent comprehensive study showed that the astrocytes in PiD are 3R-tau positive [60]. However, a few protoplasmic astrocytes in PSP also contained 3R-tau and a few astrocytes in PiD contained 4R-tau. Moreover, tau phosphorylation sites, conformational modifications, tau truncation and ubiquitination in astrocytes differed between various types of tauopathies [60]. Tau (AT8) immunoreactivity in oligodendrocytes comprises coiled bodies and globular oligodendroglial inclusions (GOIs) [33,56]. GOIs (usually >oligodendroglial nucleus, up to 15 μm) are characteristic of GGTs. Some GOIs show multiglobular appearance. It must be noted that globular inclusions (<oligodendroglial nucleus, 2 3 μm) can be seen in the white matter in PiD. Although coiled bodies are consistently 4R-tau immunoreactive, occasional GOIs in GGTs and globular inclusions in PiD contain 3R-tau as well [60] (Table 2). Characteristic neuropathological features of sporadic tauopathies The major features of tauopathies are compared in Table 3. Here the most important features are summarized. PiD Degeneration of the frontal and temporal lobes and detection of neuronal cytoplasmic Pick bodies that are 3R-tau immunoreactive are the most characteristic features (Figure 3a). Electron microscopy of Pick bodies reveals filamentous material with osmiophilic granular and vesicular structures. Tau filaments are characterized by 15 18 nm diameter straight tubules and 22 24 nm diameter twisted filaments [61,62]. Pick bodies predominate in the granule cells of the dentate gyrus, followed by the hippocampus, and cortical areas but are seen also in subcortical structures (Figure 3b). Some of the remaining neurones in the cortex are ballooned and referred to as Pick cells. Some biochemical studies suggest a significant amount of 4R-tau pathology in PiD [63]; however, the presence of 4R-tau is most likely due to the presence of concomitant neurofibrillary degeneration or rarely argyrophilic grains in the examined samples [17]. Less frequently, tau immunoreactive ramified astrocytes and rare globular inclusions (2 3 μm) in oligodendrocytes are seen. A further tau immunoreactivity (AT8) is the diffuse,

8 G. G. Kovacs Figure 2. Tau (AT8) immunoreactivity in neurons, astrocytes and oligodendrocytes. Ubi, ubiquitin or p62; NFT, neurofibrillary tangle; IR, immunoreactivity; * in some diseases (e.g. PSP) subcortical NFTs are not consistently Ubi+ve.

Neuropathology of tauopathies 9 Table 2. Definition and staining properties of major AT8 immunoreactive (IR) structures Cell type IR structure Definition 4R 3R Gallyas Biel Ubi/p62 Neurone Pretangle Diffuse fine granular staining of neuronal cytoplasm + NFT Fibrillar intracellular cytoplasmic structures + +/ * + + +/ * Pick body Cytoplasmic fibrillar spherical structures + + + + + +/ + Globular cytoplasmic structures that are various sized, and the staining pattern does not match the current definition of a Pick body Other spherical inclusions (Pick-body-like) Dystrophic neurite Rounded, oval or elongated thick profiles accumulating mostly around amyloid plaques +/ +/ + + + Threads A segment of a thin neuronal process usually associated to axons +/ +/ + +/ +/ Grains 4 9 μm spindle, coma or dot-like structures in the neuropil that are associated with dendrites + + +/ + AstroG Tufted Star-like tufts of densely packed fibres in the proximal segment of astrocytic processes + + +/ + Atsrocytic plaque Annular cluster of short stubby lesions representing the distal segments of astrocytic processes + + +/ + Thorn-shaped Thorn-shaped mostly in subpial/subependmyal and white matter location + +/ +/ +/ GAI Small globules in the astrocytic processes + +/ OligoDG Coiled body Coil-like or coma-like intracytoplasmic profiles + + +/ + GOI Globular, spherical or conical shaped structures with the diameter up to 15 μm + +/ + +/ + *Depending whether it is in subcortical structures associated with 4R tauopathy or in the cortex or in the hippocampus. AstroG, astroglia; OligoDG, oligodendroglia; Biel, Bielschowsky silver staining; Ubi, ubiquitin; +, consistently stained;, not stained; +/, variably stained. synapse-like immunostaining of the neuropil (Figure 3c) [17]. PSP Macroscopic evaluation reveals atrophy of the subthalamic nucleus and brainstem tegmentum, and depigmentation of the substantia nigra. Microscopic features comprise NFTs in subcortical structures, in particular the subthalamic nucleus, basal ganglia and brainstem, that are variably but characteristically associated with tufted astrocytes, and oligodendroglial coiled bodies, as well as threads, which are all immunoreactive for 4R-tau and negative for 3R-tau (Figure 3d f). Ultrastructural examination reveals 15 18 nm straight filaments, and NFTs show compact accumulations of 14 nm straight tubules; tubular profiles and straight filaments are seen in glial cells as well [34,61]. There is considerable clinical and pathoanatomical heterogeneity of PSP. Neuropathological hallmarks of PSP cannot predict the clinical features of Richardson s syndrome reliably [19]. Williams and co-workers have shown that the pallido-luyso-nigral system is affected early, followed by the involvement of the basal ganglia, pontine nuclei, and dentate nucleus, and later the frontal and parietal lobes, and finally other neocortical areas and cerebellar structures [64]. CBD Asymmetric focal cortical atrophy and depigmentation of the substantia nigra is a frequent macroscopic finding in CBD. According to the neuropathological criteria, Gallyas/Tau-positive lesions in CBD comprise neuronal inclusions, threads in the white and grey matter, coiled bodies and astrocytic plaques [65]. These profiles are all immunoreactive for 4R-tau and negative for 3R-tau. The minimal pathologic diagnostic features of CBD are cortical and striatal tau-positive neuronal and glial lesions, especially astrocytic plaques (Figure 3g,h) and thread-like lesions, along with neuronal loss in focal cortical regions and in the substantia nigra. Small NFTs and spherical inclusions (called also as corticobasal bodies) can be also seen (Figure 3i). Ultrastructural examination reveals 20 24 nm twisted ribbons, but tubular structures and amorphous profiles are seen in astrocytes, and twisted tubules in oligodendroglial cells [61]. Ballooned achromatic neurones in affected cortices may be noted; however, they are not highly specific for CBD. Recent studies suggest that PSP and CBD represent a disease spectrum where the

10 G. G. Kovacs Table 3. Comparison of tauopathy forms Disease Feature AD AGD CBD PSP PiD PART GGT TAG CTE Biochemistry/pathology 3R + 4R + + + + 4R>> + + + + 3R>> + Bands: 60, 64, 68, (72) kda + + + + Bands: 64, 68 kda + + + + Bands: 60, 64 kda + Cytopathology NFT: Cortex + /+ /+ /+ /+ + NFT: Subcortical /+ /+ + /+ /+ + Diffuse NC IR + + + + + + + /+ Pick body (3R+) + Spherical NCI (4R+) + + Threads in cortex + + + /+ /+ /+ /+ Threads in subcortical GM + + /+ /+ Threads in WM + + /+ /+ /+ Oligodendroglial CB + + + + /+ /+ GOI (up to 15 μm) + GAI + Ramified astrocytes+goi (<4 μm) + Tufted astrocytes + /+ /+ /+ Astrocytic plaques + /+ Thorny astrocytes (Cx/WM) * * * * * + /+ Diffuse granular astrocytic IR + /+ /+ + Astrocytic tangles + AD, Alzheimer s disease; AGD, argyrophilic grain disease; CB, coiled-body; CBD, corticobasal degeneration; DLB, dementia with Lewy bodies; PSP, progressive supranuclear palsy; PiD, Pick s disease; PART, primary age-related tauopathy (includes the formerly called neurofibrillary tangle only dementia); GGT, glial globular tauopathy; TAG, tau-astriogliopathy in the elderly; CTE, chronic traumatic encephalopathy; Cx, cortex; GOI, globular oligodendroglial inclusions; GAI, globular astroglial inclusions; IR, immunoreactivity; NC, neuronal cytoplasmic; NCI, neuronal cytoplasmic inclusion; NFT, neurofibrillary tangle; WM, white matter; *, in elderly cases may be present; +, characteristic/usually seen; /+, can be seen. depending on concomitant AD. Note that thorny astrocytes here indicate not those seen in subpial and periventricular regions but as focal accumulations in the white matter or as clusters in the gray matter. clinical presentation depends on the distribution of pathology [22]. In general, neuronal tau pathology in CBD affects mostly forebrain, while in PSP mainly hindbrain structures [66]. Despite the similarities in their genetic background and biochemical features, the distinct astroglial taupathology together with the presence of subcortical tangles in PSP allows neuropathological differentiation of CBD and PSP. This is supported by experimental observations suggesting that these two disorders may be associated with distinct tau species [67]. AGD Neuropathological features of AGD include atrophy of the ambient gyrus and the presence of argyrophilic and 4R-tau immunoreactive grains (Figure 3j) in medial temporal lobe structures that are variably associated with pretangles and oligodendroglial coiled bodies in the hippocampal and peri-amygdaloid white matter (Figure 3k). Bushy astrocytic profiles may be noted mainly in the amygdala (Figure 3k). Tolnay and Clavaguera [68] proposed that the core lesions comprise argyrophilic grains as an essential feature for the diagnosis. The number of senile plaques is low, and the Braak stages of NFTs vary between I and III. Grains that are detectable by AT8 immunostaining were originally described as argyrophilic grains, hence the name AGD [69]. Immunostaining for p62 and ubiquitin is also helpful to detect grains, although age-related tau negative neuritic profiles should be distinguished [55,70]. AGD-related pathology (grains, diffuse neuronal tau immunoreactivity and glial tau pathology) can be staged [71,72]: in stage I, the ambient gyrus and

Neuropathology of tauopathies 11 Figure 3. Histopathological findings in tauopathies (I). (a) Pick bodies (PBs) are argyrophilic using Bielschowsky staining (dentate gyrus) and immunoreactive for 3R-tau (right upper inset) and remain negative for 4R-tau (right lower inset). (b) PBs (sometimes multiple, arrowhead) in the locus coeruleus; (c) diffuse synaptic-like staining in the hippocampus using AT8 antibody; Tau (AT8) immunoreactivity in the frontal cortex (d), caudate nucleus (e) and tegmentum of the mesencephalon (f) in a representative case with progressive supranuclear palsy; Tau (AT8) immunoreactivity in the frontal cortex (g), striatum (h) and substantia nigra (i; spherical corticobasal body indicated by an arrowhead) in a representative corticobasal degeneration case; Argyrophilic grains in argyrophilic grain disease are visualized also by immunostaining for 4R-tau (j); in addition, in the amygdala bushy astrocytic profiles (k, left) and in the hippocampal white matter, oligodendroglial coiled bodies are seen (k, right); moreover, mainly cells of the granule cell layer of the dentate gyrus show diffuse cytoplasmic tau immunoreactivity (l). Bar in a represents 10 μm for a; 50μm for b and i; 200 μm for c; 100 μm for d h; and 30 μm for j l.

12 G. G. Kovacs most anterior part of CA1 is mostly affected; the amygdala and the lateral tuberal hypothalamic nucleus are mildly involved; in stage II, the CA1 is more widely involved, furthermore, the dentate gyrus (neuronal tau immunoreactivity) (Figure 3l) and presubiculum also show tau pathology; in addition in stage III the CA2/3, hypothalamic nuclei, anterior temporal, cingulate, insular and orbitofrontal cortices, accumbens nucleus and septal nuclei are affected; further expansion to the neocortex and brainstem is noted in stage IV [71]. In stage III, ballooned neurones in the amygdala and superficial spongiosis mostly in the ambient gyrus become evident. Argyrophilic grains are age associated and are frequent concomitant pathological findings in the brains of elderly individuals. It is suggested that the presence of argyrophilic grains may lower the threshold for dementia [73]. Primary age-related tauopathy This term and working classification has been only recently introduced [74]. Patients with mild-to-moderate AD-type neurofibrillary degeneration in the medial temporal lobe, but lacking Aβ plaques, have been described in several studies [74] and mostly termed NFT-dementia. Histopathological features include neurofibrillary degeneration, including ghost tangles, restricted to the hippocampus and medial temporal lobe (Figure 4a,b). These cases are usually classified as stages up to IV according to Braak staging of neurofibrillary degeneration [74]. Ultrastructural studies of sarkosyl extraction fractions reveal predominantly paired helical filaments [37]. Granule cells of the dentate gyrus and neurones of the CA4 subregion may also show tau immunoreactivity (Figure 4c), a feature, together with the ghost tangles, detected mostly in advanced Braak stages when seen as AD-related neuropathologic change. Neuronal tau pathology may be observed in the basal nucleus, in the substantia nigra, locus coeruleus and accumbens nucleus [75]. Hippocampal sclerosis may also be noted in about 10% of cases [28]. Compared with the recently suggested limbic predominant form of AD [29], the Braak NFT stage is also lower; furthermore, there are clearly fewer or no Aβ plaques in PART (NFT-dementia) [30,74]. GGTs The brain shows atrophy of the frontal and temporal lobes or precentral gyrus. This is associated with argyrophilic (Gallyas) and 4R-tau immunoreactive globular oligodendroglial and non-argyrophilic (Gallyas), 4R-tau immunoreactive globular astroglial inclusions (GOI and GAI, together termed globular glial inclusions [33] (Figure 4d,e). By electron microscopy, GOIs can be seen as granular material and haphazardly oriented filaments of 8 9 nm in diameter [38]. Gallyas staining of GOIs strongly resembles the glial cytoplasmic inclusions (GCI or Papp- Lantos body) seen in multiple system atrophy (MSA); however, in contrast to GOIs in GGT, those are α-synuclein immunoreactive [38]. Neuronal tau-pathology is mainly represented by diffuse cytoplasmic immunoreactivity, globular or small tangle-like inclusions (Figure 4f), which are composed mainly of the 4R-tau isoform. The anatomical distribution of tau pathology correlates with the predominant clinical symptoms. Recent consensus recommendations proposed at least three subtypes [33]. Cases with predominantly frontotemporal involvement and prominent GOIs in the white matter are termed type I. Type II cases show an anatomically more restricted involvement of the motor cortex and corticospinal tract (while in other regions PSP-like morphological features may be seen) with GOIs and GAIs. Those with frontotemporal, motor cortex and corticospinal tract involvement associated with an abundance of GAIs in cortical areas are termed as type III. Although GGTs are thought to be rare, recent studies showed that these forms may be observed in the elderly associated also with AD-type pathology [16,18]. Overlapping features of primary tauopathies Presence of argyrophilic grains in the limbic system is frequent in PSP, CBD, PART or advanced stages of AD. Detection of neuronal tau immunoreactivity in the CA2/3 subregion with sparing of the CA1 [76], as well as in the granule cells in the dentate gyrus, is frequently seen in 4R tauopathies. These features can be helpful, for example, to raise the suspicion of AGD as a concomitant pathology in AD cases. Indeed, concomitant AGD influences the distribution-pattern of tau immunoreactivity in the hippocampus [77]. Further forms of tauopathy Many other diseases with diverse aetiology may be associated with tau pathology (summarized in Table 4 and see also Figure 4g,h). There are occasional reports on cases of tauopathy where the neuropathological phenotype is not

Neuropathology of tauopathies 13 Figure 4. Histopathological findings in tauopathies (II). (a) Overview of tau immunoreactivity in the hippocampus (note the brown coloured pathological deposits) in primary age-related tauopathy (PART, formerly also called as NFT-dementia); (b) prominent neurofibrillary tangles in the CA1 subregion, but also in the CA4 subregion and in the dentate gyrus granule cells (c) in PART. (d) In the hippocampus, the white matter is involved prominently in globular glial tauopathy (GGT Type I) (note the brown coloured pathological deposits; compare with a); in the region of the basal ganglia (e), the frontobasal white matter and internal capsule show the pathological deposits. Note the distribution of tau pathology in the cortico-subcortical junction in GGT (f; arrowhead shows neuronal tau-immunoreactive profile; dashed line indicates the border of the white matter). Fine neuritic type of tau immunoreactivity in the brain of a representative case with sporadic Creutzfeldt Jakob disease (g); in genetic Creutzfeldt Jakob disease (here E200K mutation) some cases show much more prominent neuritic and thread-like pathology (here in the basal ganglia, h). Accentuation of tau pathology around small cerebral vessels at the depths of the cerebral sulci (arrowhead) and in subpial areas in the frontal cortex (i) in early chronic traumatic encephalopathy. Patterns of tau pathology in the elderly brain include prominent thorny astrocytic profiles in the dentate gyrus (arrowhead) and CA subregions (j); clusters of thorny astrocytes in the basal ganglia (here nucleus accumbens, k) and also in some cases in the substantia nigra (l). Bar in a represents 300 μm for a and d; 100 μm for b, c and f; 200 μm for e and j, 30μm for g, h and k, 150 μm for i and 50 μm for l.

14 G. G. Kovacs Table 4. Overview of tau pathologies associated with diverse disorders Disorder Tau pathology Ref. AD and DS Neurofibrillary degeneration, neuropil threads and dystrophic neurites Lewy body disorders Frequent co-occurrence with tauopathies Tuft-shaped astrocytes in a subgroup of DLB patients [78] LRRK2 and α-synuclein gene mutations may be associated with tauopathy [79 81] C9orf72 repeat More neurofibrillary tangles and higher tau burden compared with FTLD-TDP (GRN), however similar [82] mutation to that seen in sporadic FTLD cases with TDP-43 pathology Associated with the MAPT sequence variation (Ala239Thr): Tauopathy comprised 3R-tau [83] immunoreactive Pick bodies and 4R-tau immunoreactive glial tau pathology Prion diseases Small neuritic profiles in all prion disease types; Larger dystrophic neurites and neuritic profiles around [84] amyloid plaques (GSS, vcjd) Widespread neurofibrillary degeneration in several subcortical, allo- and neocortical anatomical [85] regions: GSS Atypical forms of tauopathies in genetic CJD [84,86] DNTC Co-occurrence of neurofibrillary tangles and Fahr s type calcification [87,88] Coiled bodies, clusters of thorny and tufted astrocytes may be seen [89] Co-occurrence of α-synuclein and TDP-43 proteinopathy and tauopathy [90] FBD and FDD Prominent ABri or ADan amyloid deposition is associated with tau positive NFTs, neuropil threads and [91] dystrophic neurites NBIA NFTs and threads in PANK2 gene mutation [92] Threads, pretangles, NFTs in PLA2G6 gene mutations [93] NPDis type C NFTs also in brainstem and spinal cord, cerebellum [94] PEP Globose NFTs mainly in brainstem nuclei, and NFTs also in the hippocampus, temporal, frontal and [95] insular cortex. NFT pathology comprises both 3R- and 4R-tau. [96] ParkDem/Guam NFTs in the hippocampus, temporal and frontal cortices, basal ganglia, thalamus, substantia nigra, [97] superior central nucleus and locus coeruleus Tau astrogliopathy and oligodendroglial coiled bodies The biochemical signature is similar to AD Co-occurrence of α-synuclein and TDP-43 proteinopathy and tauopathy SSPE NFTs as seen in AD and glial fibrillary tangles [98] SLC9A6/mental The neuropathological phenotype is reminiscent of CBD [99] retardation Myotonic dystrophy NFTs in young age and a tau profile characterized by a strong band at 60 kda and to a lesser extent at [7] 64 and 69 kda CTX Early onset limbic 4R tauopathy with AGD-like features [100] TARDBP mutation p.ile383val is associated with complex tauopathy [101] Tau astrogliopathy in For details see text the elderly CTE For details see text AD, Alzheimer s disease; DS, Down syndrome; FTLD, frontotemporal lobar degeneration; GRN, progranulin gene; GSS, Gerstmann-Sträussler- Scheinker disease; CJD, Creutzfeldt-Jakob disease; vcjd, variant CJD; DNTC, diffuse neurofibrillary tangles with calcification; FBD and FDD, familial British and Danish dementia; NBIA, neurodegeneration with brain iron accumulation; NPDis, Niemann-Pick disease; PEP, postencephalitic parkinsonism; ParkDem, Parkinsonism-dementia complex of Guam; SSPE, subacute sclerosing panencephalitis; CTX, cerebrotendinous xanthomatosis; CTE, chronic traumatic encephalopathy. compatible with well-known entities. In some of these reports, genetic analysis is missing. Recently, a 4R predominant tauopathy was reported [102]. This was associated with hippocampal sclerosis and numerous intracytoplasmic inclusions in the dentate granule cells. The slightly basophilic, inclusions show argyrophilia with Bodian s and Gallyas Braak method and are 4R- but not 3R-tau immunoreactive. Chronic traumatic encephalopathy (CTE) CTE is a progressive neurodegeneration triggered by repetitive mild traumatic injury. It usually begins 8 10 years after the repetitive trauma and is characterized by widespread deposition of hyperphosphorylated tau [103]. Western blot shows that soluble and insoluble tau is indistinguishable from that in AD [104]. Tau pathology

Neuropathology of tauopathies 15 includes NFTs and astrocytic fibrillary deposits. CTE is distinguished from other tauopathies by a distinctive topographic and cellular pattern of tau pathology [105]. There is an accentuation of tau pathology around small cerebral vessels and at the depths of the cerebral sulci (Figure 4i). Astrocytic tau pathology may be prominent in subpial and periventricular areas. Tau-positive astrocytes in CTE may form astrocytic tangles [105]. A recent comprehensive study on individuals with CTE recognized four stages [106] distinguished by the progressive involvement of the frontal, temporal and parietal cortices, with relative sparing of the calcarine cortex. In parallel, neurofibrillary degeneration appears increasingly in subcortical structures and brainstem nuclei. Rarely CTE type pathology may be associated with features of primary tauopathies, like PSP, giving a challenge for the diagnostic workup [107]. Overview of tau pathologies in the ageing brain In the ageing brain, the most frequent alteration is neurofibrillary degeneration with or without senile plaques (i.e. PART) and argyrophilic grains. In addition, PSP-like pathology is also seen in the elderly even without clinical evidence of PSP [14 16]. Widespread application of phospho-tau (AT8) immunohistochemistry revealed further tau pathologies. Unfortunately, there is a lack of consensus as to how these can be grouped or their clinical relevance. However, tau astrogliopathy is a common feature. Thorn-shaped astrocytes are regarded as nonspecific secondarily induced tau-positive astrocytes [108]. A high prevalence of thorn-shaped astrocytes has been reported in the human medial temporal lobe [109] mainly in periventricular and subpial localizations. A systematic study in AD found approximately 30% frequency that increased with age [110]. It was concluded that thorny astrocytes are independent from AD pathology. Munoz and co-workers described argyrophilic thorny astrocyte clusters (termed ATACs) in the fronto-temporo-parietal cortex and subcortical white matter in a cohort of patients with possible progressive aphasia and AD pathology [111]. Further cases of tau-astrogliopathies have been reported in a subset of patients with dementia in the elderly [58] and characterized further in the context of the spectrum of neuropathological alterations in elderly brains [16]. In that study, four groups were distinguished based on the distribution of tau astrogliopathy: restricted to the medial temporal lobe or expanding into subcortical structures and the substantia nigra or even into the medulla oblongata [16] (Figure 4j l). Tau astrogliopathy was not associated with NFTs and was not accentuated in the depth of sulci as in CTE. The extent of tau pathology correlated with the clinical symptoms. Interestingly, some of these cases of the elderly frequently showed TDP-43 pathology expanding beyond the medial temporal lobe; in this aspect, it is intriguing to find reports of similar tau pathology in TARDBP mutation TDP-43 proteinopathies [101]. The morphology of AT8 immunoreactive astrocytes ranged from fine granular immunoreactivity in the processes (Gallyas negative) to those with plump perikarya and thorny appearance (partly Gallyas positive). Importantly, the thorny astrocytes often appeared in clusters in the elderly brain as reported by Munoz in their series of AD cases [111] but also showing massive focal accumulations in the white matter. In this respect, it is of note that subcortical white matter astrocytic tau pathology was reported in two elderly patients, where CBD was considered [112]. Finally, tuft-shaped astrocytes were reported in dementia with Lewy bodies [78] and also in the occipito-temporal gyrus in fewer aged brains [113]. Furthermore, Beach et al. reported a series of cases with hippocampal sclerosis and tau-astrogliopathy described as tufted, thorned, fibrous and protoplasmic forms [114]. In summary, these reports show considerable overlaps. A recent study with mathematical analysis of the distribution in hippocampal subregions [77], as well as molecular pathological evaluation of glial tau pathology [60], supports the concept that those tau astrogliopathies occurring in the elderly that are not restricted to the subpial and periventricular regions may be indeed distinct entities. Thus, the different descriptions most likely represent a spectrum or stages of similar conditions described with different terms. In spite of the presence of 4R-tau immunoreactivity in the astroglial cells, biochemical investigations show a mixture of 3R and 4R isoforms, most likely due to the concomitant AD-related neuropathologic alterations that contaminate the homogenates prepared for immunoblotting [58]. Consensus is very much needed, and guidelines are being developed for further research studies. Practical diagnostic approach to tauopathies The first step should be the evaluation of histopathological alterations based on basic nuclear and myelin stains. This should include documentation of vascular

16 G. G. Kovacs

Neuropathology of tauopathies 17 Figure 5. Algorithm of a cost-effective diagnostic strategy to evaluate tauopathies. Following the mapping of neuronal loss and gliosis, screening for tau pathologies can be performed by immunostaining blocks of the basal ganglia (BGG; caudate and accumbens nucleus, putamen), amygdala and hippocampus at the level of the lateral geniculate body, containing also the entorhinal cortex and inferior temporal cortex, for tau (AT8 immunohistochemistry). In the BGG neurofibrillary tangles (NFTs) and glial pathology should be evaluated. Tau immunoreactivity in the glia should be stratified as astro- or oligodendroglial, and the most relevant astroglial (AG, including TA, tufted astrocyte; AP, astrocytic plaque; GAI, globular astroglial inclusion; and other, like clusters of thorny astrocytes or fine granular tau immunoreactivity in the astroglial processes) and oligodendroglial (OG, including CB, coiled body; GOI, globular oligodendroglial inclusion) morphologies should be recognized. In the amygdala, the evaluation should focus on the presence of astroglial (clusters of thorny astrocytes or fine granular tau immunoreactivity in the astroglial processes), pretangle (PT) and dendritic grain-type (GR) neuron-related tau immunoreactivities. In the hippocampus several subregions should be evaluated. In particular, grains and NFTs and astroglial pathology (clusters of thorny astrocytes or fine granular tau immunoreactivity in the astroglial processes) should be noted in the inferior temporal and entorhinal cortex (EN), subiculum and CA1 subregion. Pretangles, spherical bodies (SB; 3R or 4R immunoreactive) and astroglial (thorn-shaped) are important to recognize in the dentate gyrus (DG). Finally, the white matter may also show thorny astrocytes or oligodendroglial tau immunoreactivity. Constellations of these tau immunoreactivities may be suggestive of certain tauopathies, however, for a correct characterization, including the detection of rare variants, systematic mapping of further anatomical regions (summarized in the lower part of the image as grey boxes) is warranted. In all evaluated regions care should be taken to note dystrophic neurites that are associated with amyloid plaques suggestive of AD-related neuropathologic change. Unexpected amount of NFT-like changes and astrocytic tau pathology, or enhancement of these in the depth of the sulci (i.e. in the block of the hippocampus containing the sulcus collateralis) should also prompt the consideration of chronic traumatic encephalopathy. Dotted line indicates overlap between disorders. Further abbreviations: AGD, argyrophilic grain disease; CBD, corticobasal degeneration; GGT, globular glial tauopathy; PART, primary age-related tauopathy (formerly called also as NFT-dementia); PID, Pick s disease; PSP, progressive supranuclear palsy; TAG, tau-astrogliopathy in the ageing brain; Uncl, unclassifiable tauopathy. Anatomical regions are the following: TE, temporal cortex; CI, anterior cingulate; FR, frontal cortex; MOT, motor cortex; PA, parietal cortex; GP, globus pallidus; TH/ST, thalamus and subthalamic nucleus; ME, mesencephalon including tectum, tegmentum and substantia nigra; PO, pons; MO, medulla oblongata; CB/DN, cerebellar cortex, white matter and dentate nucleus. lesions, neuronal loss and gliosis (complemented by immunostaining for glial fibrillary acidic protein,gfap), neuronal changes like basophilic fibrillar structures and ballooned neurones (immunostaining for α-b-crystallin can be helpful). This is followed by the evaluation of phospho-tau (for diagnostics AT8 is recommended) immunostained sections: evaluation and classification of the type of cytopathology, including characterization of the affected cell types i.e. neuronal, mixed neuronal/glial or glial predominant. It is helpful to score immunoreactivities and to perform anatomical mapping of immunostaining patterns. The anatomical distribution of the tau pathology (hippocampus, limbic system or neocortical predominant vs. basal ganglia, thalamus, subthalamus or brainstem predominant) and involvement of the white matter should be noted. AT8 and other phospho-tau antibodies also label those pathological structures that are not in a fibrillar form or ubiquitinated. To clarify this, additional immunostaining (i.e. AT100, ubiquitin, p62) and Gallyas silver staining can be performed. Finally, immunoblotting investigations or ultrastuctural evaluation of filament preparations can be done and consultation for genetic analysis of the MAPT gene can be sought. Multidisciplinary comparison is very helpful for the final interpretation. Considering the anatomical involvement of primary tauopathies, including rare variants and differences in the burden of pathologies, screening for tauopathies can be achieved by immunostaining (AT8) the block representing the hippocampus at the level of the lateral geniculate body, amygdala and basal ganglia. Recognition of subregional/ layer involvement and cytopathological constellations can be helpful to suggest different forms of tauopathy, but further regions need to be evaluated for full characterization of a disorder [115]. Indeed, mathematical analysis of AT8 immunoreactivity patterns in 24 hippocampal subregions/layers revealed disease-specific hotspots and regional selective vulnerability [77]. These hotspots can be shifted in cases with concomitant tauopathies (i.e. AGD with AD or other tauopathy). Thus, involvement of the hippocampus in PSP, CBD, PiD and GGT can be distinguished from early AD-related changes (i.e. Braak stage related tau pathology in the hippocampus) [77]. Knowledge of these vulnerability patterns is important as when one screens the hippocampus, tau pathology can be interpreted as related to early Braak stages and further systematic evaluation is then omitted. This could potentially lead to less correct evaluation of the frequencies of tauopathies in autopsy surveys. A strategy for the diagnostic evaluation of tauopathies is summarized in Figure 5. Mutation in the MAPT gene should be suspected in all cases, in particular when abundant tau pathology is observed.