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Neuropathology 2014; 34, 555–570

doi:10.1111/neup.12143

Sympos i um : D e fi n i t i o n a n d D i ff e r e n t i a l s – H o w t o D isti ngui s h D i s e a s e - S p e c i fi c C h a n g e s o n M i c r o s c o p y

Astrocytic inclusions in progressive supranuclear palsy and corticobasal degeneration Mari Yoshida Institute for Medical Science of Aging, Aichi Medical University, Aichi, Japan

Tufted astrocytes (TAs) in progressive supranuclear palsy (PSP) and astrocytic plaques (APs) in corticobasal degeneration (CBD) have been regarded as the pathological hallmarks of major sporadic 4-repeat tauopathies. To better define the astrocytic inclusions in PSP and CBD and to outline the pathological features of each disease, we reviewed 95 PSP cases and 30 CBD cases that were confirmed at autopsy. TAs exhibit a radial arrangement of thin, long, branching accumulated tau protein from the cytoplasm to the proximal processes of astrocytes. APs show a corona-like arrangement of tau aggregates in the distal portions of astrocytic processes and are composed of fuzzy, short processes. Immunoelectron microscopic examination using quantum dot nanocrystals revealed filamentous tau accumulation of APs located in the immediate vicinity of the synaptic structures, which suggested synaptic dysfunction by APs. The pathological subtypes of PSP and CBD have been proposed to ensure that the clinical phenotypes are in accordance with the pathological distribution and degenerative changes. The pathological features of PSP are divided into 3 representative subtypes: typical PSP type, pallido-nigro-luysian type (PNL type), and CBD-like type. CBD is divided into three pathological subtypes: typical CBD type, basal ganglia- predominant type, and PSP-like type. TAs are found exclusively in PSP, while APs are exclusive to CBD, regardless of the pathological subtypes, although some morphological variations exist, especially with regard to TAs. The overlap of the pathological distribution of PSP and CBD makes their clinical diagnosis complicated, although the presence of TAs and

Correspondence: Yoshida Mari, MD, PhD, Institute for Medical Science of Aging, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan. Email: [email protected] Received 16 April 2014; revised 15 June 2014 and accepted 15 June 2014.

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APs differentiate these two diseases. The characteristics of tau accumulation in both neurons and glia suggest a different underlying mechanism with regard to the sites of tau aggregation and fibril formation between PSP and CBD: proximal-dominant aggregation of TAs and formation of filamentous NFTs in PSP in contrast to the distaldominant aggregation of APs and formation of less filamentous pretangles in CBD. Key words: astrocytic plaque, corticobasal degeneration, fibril formation, progressive supranuclear palsy, tufted astrocyte.

INTRODUCTION Progressive supranuclear palsy (PSP)1 and corticobasal degeneration (CBD)2 have been regarded as distinct clinicopathological entities with hyperphosphorylated four repeat (4R) tau aggregation in neurons and glia, although the recent recognitions of many clinical similarities have increasingly raised more difficulties in the clinical diagnosis of these two disorders. However, microscopic cellular tau pathology has been used to distinguish PSP from CBD.3–5 PSP is defined primarily by tau-positive neurofibrillary tangles (NFTs), coiled bodies, threads, and tufted astrocytes, in contrast to the ballooned neurons, pretangles, threads, and astrocytic plaques that are characteristic of CBD (Table 1). Because PSP and CBD frequently present similar pathological distributions (Table 1, Fig. 1), a pathological diagnosis may be difficult without the discrimination of abnormal tau inclusions and particularly of the most characteristic and obvious tau morphology, that of astrocytic inclusions.6 Therefore, it is important to reevaluate and differentiate between tufted astrocytes (TAs) and astrocytic plaques (APs) and to discuss the pathogenesis of these types of inclusions. To address these issues, we reviewed the morphology and differential distribution of

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Table 1 Diagnostic pathological findings in progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) PSP Lesion Neuronal loss & gliosis

Affected cortices (variable) Globus pallidus Subthalamic nucleus Substantia nigra Brainstem tegmentum Dentate nucleus Pons and medulla (variable)

Ballooned or achromatic neurons Gallyas/4R-tau positive lesions Neuronal inclusions

Threads and coiled bodies

Astrocytic inclusions

CBD Distribution Affected cortices/subcortical white matter (gliosis) Globus pallidus (variable) Subthalamic nucleus (variable) Substantia nigra Striatum (caudate and putamen) (gliosis) Brainstem tegmentum (variable) Dentate nucleus (variable) Pons and medulla (variable) Affected cortices

NFTs > Pretangles Substantia nigra, oculomotor complex, locus ceruleus, pons, brainstem nuclei, dentate nucleus, globus pallidus, subthalamic nucleus Striatum, thalamus, basal nucleus of Meynert Affected Cortices (variable) Spinal cord Threads and coiled bodies Brainstem, cerebellar white matter, globus pallidus, subthalamic nucleus, striatum, thalamus, gray matter and white matter, spinal cord Tufted astrocytes Affected cortices, striatum, brainstem

Pretangles >> NFTs Affected cortices, substantia nigra, globus pallidus, subthalamic nucleus

Striatum, thalamus, basal nucleus of Meynert Brainstem nuclei and dentate nucleus Spinal cord Threads >> coiled bodies Subcortical white matter and gray matter, globus pallidus, subthalamic nucleus, striatum, thalamus, brainstem, spinal cord Astrocytic plaques Affected cortices, striatum

4R-tau, 4 repeat tau; NFTs, neurofibrillary tangles.

pathologic lesions of 95 neuropathologically confirmed PSP cases and 30 CBD cases that were registered at the Institute for Medical Science of Aging, Aichi Medical University. Our focus was on the pathogenesis of astrocytic inclusions, their disease specificity, and their morphological variations.

PROGRESSIVE SUPRANUCLEAR PALSY (PSP) PSP is a progressive neurodegenerative disorders,described by Steele, Richardson and Olszewski in 1964.1 It is the second most common form of parkinsonism after Parkinson’s disease. Clinical features include abnormal gait, and postural instability, and recurrent falls, supranuclear ophthalmoparesis, cognitive and behavioral changes, pseudobulbar features and dystonia.

Clinical aspects As noted previously, pathologically confirmed cases of PSP have exhibited some variation of the clinical and pathological features. Therefore, clinical subtypes were proposed to classify PSP: PSP-Richardson, PSP-parkinsonism (PSPP),7,8 PSP-pure akinesia with gait freezing (PSP-PAGF),9 PSP-primary progressive aphasia,10,11 and PSP-predominant

cerebellar involvement (PSP-C).12,13 The PSP-Richardson type is the prototypical form of PSP that is defined by early falls, early cognitive dysfunction, abnormalities of gaze and postural instabilities. PSP-P represents asymmetric onset, tremor, early bradykinesia, non-axial dystonia and a response to levodopa. Individuals with PSP-PAGF present with the gradual onset of freezing of gait or speech, absent limb rigidity and tremor, a lack of sustained response to levodopa, and no dementia or ophthalmoplegia in the first 5 years of disease. PSP-primary progressive aphasia is defined by the presence of primary progressive aphasia, or progressive nonfluent aphasia. Individuals with PSP-C develop cerebellar-predominant ataxia as the initial and principal symptom.

Neuropathology Neuropathological diagnostic criteria The pathological criteria for the diagnosis of PSP are well established and include specific neuronal loss with gliosis and neurofibrillary tangles (NFTs) in the subcortical and brainstem nuclei and in the cerebellar dentate nucleus with the pathological accumulation of abnormally phosphorylated microtubule-associated protein tau into filamentous deposits.14 The NINDS diagnostic criteria for PSP and related disorders are pertinent for typical PSP, © 2014 Japanese Society of Neuropathology

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Fig. 1 The group of pathological subtypes in PSP (A) and CBD (B). (A) The pathological subtypes in PSP is generally divided into three representative types: typical PSP type, pallido-nigro-luysian type (PNL type), and CBD-like type, according to the distribution of the lesions and the severity. The spinocerebellar degeneration (SCD)-like type is associated with severe degeneration of the dentate nucleus, superior cerebellar peduncles, cerebellar cortex, white matter and pontine tegmentum and base, which are frequently associated with frontal involvement. The PNL type shows relatively restricted changes in pallido-nigro-luysian lesions. The CBD-like type is accompanied by more severe, asymmetrical cortical changes and a variable degeneration of the basal ganglia, brainstem and cerebellar dentate nucleus. (B) The pathological subtypes of CBD is generally divided into three representative types: typical CBD type, basal ganglia-predominant type, and PSP-like type, according to the distribution of the lesions and the severity. The typical CBD type shows dominant cortical involvement with laterality in the posterior frontoparietal or perisylvian areas. Some cases exhibit anterior frontal-dominant cortical degeneration, such as frontotemporal lobar degeneration. The basal ganglia-predominant type reveals severe involvement of the pallidum and subthalamic nucleus, with relatively mild cortical degeneration without distinct laterality. The PSP-like type shows severe degeneration of the brainstem and dentate nucleus similar to that seen in PSP, in addition to the variable cortical involvement.

which conforms to the original description, and atypical PSP, which consists of histologic variants where the severity or distribution of abnormalities, or both, deviate from the typical pattern; these criteria are also relevant for combined PSP, in which typical PSP is accompanied by concomitant infarcts in the brainstem, basal ganglia, or both.3,12 Micro© 2014 Japanese Society of Neuropathology

scopic findings include a high density of NFTs and neuropil threads in at least three of the following areas: the pallidum, subthalamic nucleus, substantia nigra, or pons. In addition, a low to high density of NFTs or neuropil threads is found in at least three of the following areas: the striatum, oculomotor complex, medulla, or dentate nucleus. A clinical history

558 that is compatible with PSP is also required for diagnosis according to this set of criteria. These criteria have come to define the typical clinicopathological PSP cases. However, based on these criteria, it was recommended that atypical PSP should be excluded as a PSP subtype because further neuropathological studies of this entity were needed.

Neuropathological reevaluation of PSP cases Among 95 pathologically confirmed PSP cases, 25 cases were associated with other significant diseases. Two of these cases were associated with Alzheimer’s disease, 12 with Parkinson’s disease or dementia with Lewy bodies (DLB), 1 with multiple system atrophy, 1 with SCA6 and DLB, 1 with amyotrophic lateral sclerosis, 1 with traumatic brain injury, 4 with cerebrovascular disease, 1 with glioblastoma, and 2 cases were without detailed information. With the exception of these 25 cases, 70 cases were analyzed and had the following characteristics: a mean age at onset of 67 years (range 39–92 years), a mean disease duration of 8 years (range 1–28 years), and a mean age at death of 75 years (range 49–106 years). PSP is a sporadic disease, although approximately 7% of affected individuals have a family history of neurological disorders, including parkinsonism or dementia.

M Yoshida neuronal loss and gliosis, while the putamen and the caudate show mild gliosis. The most affected nuclei are the globus pallidus, subthalamic nucleus and substantia nigra. The affected regions of the brainstem are as follows: midbrain tegmentum including the superior colliculus, periaqueductal gray matter, oculomotor nuclei, locus ceruleus, pontine tegmentum, pontine nuclei, medullary tegmentum and the inferior olivary nucleus. The dentate nucleus usually exhibits grumose degeneration. The superior cerebellar peduncles are atrophic, and the cerebellar cortex may show mild loss of Purkinje cells with mild atrophy of the white matter. The medullary tegmentum may be atrophic with myelin pallor. The cerebral cortices show mild gliosis especially in the premotor and precentral gyrus in the convexity. The spinal cord, especially the cervical segment, is usually involved, particularly in the medial division of the anterior horn and intermediate gray matter.15–17 Transverse sections of the spinal cord often show myelin pallor in the anterolateral funiculus in the cervical and thoracic segments. Immunohistochemistry for phosphorylated tau or modified Gallyas silver staining reveals NFTs, pretangles in neurons, tufted astrocytes, coiled bodies in oligodendrocytes, and threads (Table 1, Fig. 3).

Tufted astrocytes Macroscopic and microscopic findings The macroscopic examination of the brain in typical PSP reveals mild frontal atrophy including precentral gyrus, particularly in the convexity (Fig. 2A). The brainstem and cerebellum are mildly atrophic. The globus pallidus and subthalamic nucleus usually show a brownish atrophy. The third ventricle may be enlarged. The tegmentum of the midbrain and pons also shows atrophy. The substantia nigra shows discolored, while the locus ceruleus is often relatively preserved. The cerebellar dentate nucleus, and the superior cerebellar peduncle are atrophic. The microscopic findings indicate neuronal loss and gliosis with NFTs, which appear globose in appearance, in the basal ganglia, thalamus, brainstem, and cerebellum (Table 1, Fig. 3). The thalamus has mild to moderate

TAs are defined as radial arrangements of thin and long branching fibers without collaterals that course continuously through the cytoplasm to the distal processes of astrocytes (Fig. 3d–j,m,n).6,18 “Tufts of abnormal fiber,” as described in PSP by Hauw et al.,19 is the root of the nomenclature for “tufted astrocytes,” although their cellular characterization was not mentioned in their study. Tufted astrocytes were described by Hauw et al.19 as star-like tufts of fibers devoid of degenerative features and without amyloid cores that are clearly distinguishable with Bodian stain as well as with tau immunocytochemistry. The astrocytic nature of the cells that contain the tufted-type inclusions was confirmed by the double-labeling of sections with antibodies to GFAP, CD44 and abnormal tau.20–22 Cytoplasmic staining is usually not conspicuous within TAs

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Fig. 2 Macroscopic findings of typical PSP type (A), pallido-nigro-luysian (PNL)- type (B) and CBD-like type (C). (A) Macroscopic findings in coronal sections of typical PSP show mild frontal atrophy in the convexity (a), atrophy of the pallidum and subthalamic nucleus (a, arrow), atrophy of the brainstem tegmentum (b), loss of pigment in the substantia nigra (b, arrow) with preservation of pigment in the locus ceruleus, and atrophy of the cerebellar dentate nucleus (c). Bar = 2 cm. (B) Macroscopic findings in PNL-type PSP reveal severe atrophy of the pallidum and the subthalamic nucleus (a) and depigmentation of the substantia nigra (b), with relative preservation of the brainstem tegmentum (b) and cerebellar dentate nucleus (c). The PNL-type sometimes shows atypical TAs, which demonstrates proximal dominant tau accumulations (d, e). d, Gallyas silver stain; e, AT8 immunohistochemistry; a, b, c, bar = 2 cm; e, bar = 10 μm. (C) Macroscopic findings in CBD-like type PSP indicate left side-predominant degeneration of the cerebral cortices and the basal ganglia (a–d) with relatively mild atrophy of the brainstem (e). Microscopically typical TAs are observed in the basal ganglia and cerebral cortices (f, g). a, c, Klüver-Barrera stain; b, d, Holzer stain; f, Gallyas silver stain; g, AT8 immunohistochemistry; bar = 10 μm.

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Fig. 3 Major microscopic findings in PSP. Microscopic findings include globose-type neurofibrillary tangles visualized with H&E stain (a), Bodian stain (b), and phosphorylated tau immunohistochemistry (c). Typical TAs have phosphorylated tau (d, g, j), 4 repeat tau (e, h) and Gallyas-positive (f, i) radiating proximal branches with variable cytoplasmic tau accumulation. Differential structures are oligodendroglial coiled bodies and threads (k) and tau accumulation in senile plaques (l),and globular glial inclusions in globular glial tauopathies (m).Double immunostaining for AT8 (brown) and GFAP (red) in TAs shows tau-positive radiating filamentous processes from the GFAP-positive cytoplasm (n) or more abundant tau accumulation in the proximal portion in the cytoplasm (o). a, H&E stain; b, Bodian stain; f, i, Gallyas silver stain; c, d, g, i–m, AT8 immunohistochemistry; e, h, RD4 immunohistochemistry; n, o, double immunostaining for AT8 (brown) and GFAP (red). Bar = 10 μm. ◀

after either Gallyas-Braak (GB) silver stain or anti-tau immunohistochemistry; however, upon careful examination, some TAs revealed positive staining in the perikaryon after anti-tau immunohistochemistry (Fig. 3g). Electron microscopy demonstrated that astrocytes with abundant glial filaments formed loose bundles of straight 15 nm tubules in the perikaryon.23 Fibers with a diameter of approximately 20–25 nm were also described.21 Immunoelectron microscopy recognized TAs as central nucleus surrounded by radiating AT8-immunoreactive processes.24 TAs are commonly found in the premotor, supplement, and motor cortex in the convex areas, the basal ganglia, thalamus, and brainstem tegmentum, and are recognized as a distinctive pathological marker of PSP.25 With regard to disease specificity, it is strongly believed that the 4R tau-positive TAs are more common in PSP. It is probable that tufted astrocytes are the pathognomonic hallmark of PSP.6,25,26

Correlation between TAs and clinicopathological features Based on the distribution of neuronal loss with gliosis and tau positive neuronal and glial inclusions, the pathological topography of 68 out of 70 PSP cases (except for 2 cases within one year’s disease duration), were divided grossly into 3 representative subtypes: typical PSP type, pallidonigro-luysian type (PNL-type), and CBD-like type (Fig. 1A,2). The typical PSP type (50 out of 68 cases, 73%) represents the prototypical pathological distribution, which involves the pallidum, subthalamic nucleus, substantia nigra, brainstem tegmentum, cerebellar dentate nucleus, thalamus and the cerebral cortices to a mild or moderate degree (Fig. 2A). SCD-like type (11 cases, 16%) involves severe degeneration of the dentate nucleus and atrophy of the pontocerebellum with tau accumulation (Fig. 1A). Because the SCD-like type normally develops into a typical PSP type, this type seems to be of similar etiology as the typical PSP type. The PNL-type (12 cases, 18%) demonstrates lesions that are relatively confined to the pallidum, subthalamic nucleus and substantia nigra (Fig. 1A,2B). CBD-like type (6 cases, 9%) reveals prominent asymmetrical cortical involvement that is associated with mild to moderate involvement of the brainstem and the cerebellar dentate nucleus (Fig. 1A,2C). © 2014 Japanese Society of Neuropathology

The pathology of the different subtypes wholly corresponds to the clinical phenotypes: pathologically typical PSP type corresponds to PSP-Richardson, pathological PNLA-types to PSP-PAGF or some PSP-P clinical phenotypes, and CBD-like type to PSP-corticobasal syndrome (CBS).27 Therefore, the clinical phenotypes depend on the topographical distribution and the degree of degeneration. Furthermore transitional pathology also exists among those pathological subtypes in their distribution and severity. In all subtypes,TAs are almost always invariably present. In the typical PSP type, TAs are usually prominent in the frontal cortices and in the striatum.6 In the CBD-like type, more prominent TAs are seen in the affected cerebral cortices. In the PNLA type, typical TAs are less prominent, and atypical astrocytic tau inclusions are sometimes observed (Fig. 2B). These astrocytes in the striatum develop marked proximal dominant tau accumulation, but these atypical astrocytic tau inclusions are always accompanied by a few typical TAs (Fig. 3d–f).TAs in the cerebral cortices develop alongside NFT formations and other glial structures such as coiled bodies and threads, although TAs are already found early in PSP cases (within one year) and are even found in cases without formation of significant NFTs or other glial inclusions. Therefore, TAs do not necessarily accompany neuronal loss and reactive gliosis, but rather, they reflect intrinsic tau aggregation in astrocytes.28 Differential structures are tau-positive dystrophic neurites that surround senile plaques, thorn-shaped astrocytes, astrocytic plaques, coiled bodies, threads, and globular glial inclusions in globular glial tauopathies,29 although these structures are generally well-differentiated based on each of their characteristic distributions and morphology, or by using immunohistochemistry to detect tau or Aβ (Fig. 3k–o).

CORTICOBASAL DEGENERATION (CBD) CBD is a rare, progressive neurological disorder characterized by widespread hyperphosphorylated 4R tau pathology in neurons and glia in both cortical and subcortical areas.4 The clinical presentation originally described by Rebeiz et al. involves asymmetric motor dysfunction including prominent involuntary movements.2 Additional reports

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have described similar patients who, in addition, demonstrated supranuclear gaze palsy and Parkinsonian features.27,30–32 Although the combination of asymmetric motor disturbances with cortical sensory loss and apraxia without marked cognitive dysfunction has been thought to be specific for CBD, neuropathologically documented presentations as PSP and Parkinson’s disease make antemortem diagnosis of CBD difficult.33–35

CLINICAL ASPECTS In light of advances in the understanding of CBD, new guidelines have recently proposed 4 CBD phenotypes: CBS, which presents as an asymmetric movement disorder combined with lateralized higher cortical features, frontal behavioral-spatial syndrome (FBS), a nonfluent/ agrammatic variant of primary progressive aphasia (naPPA), and progressive supranuclear palsy syndrome (PSPS).36

Neuropathology Neuropathological diagnostic criteria Due to the uncertainty and heterogeneity of the clinical criteria for the diagnosis of CBD, neuropathological examination is the gold standard used to make a definitive diagnosis. Clear pathological criteria have been proposed that were not adversely influenced by clinical information.5 These criteria emphasize tau-immunoreactive lesions in neurons, glia, and cell processes in the neuropathologic diagnosis of CBD, although cortical atrophy, ballooned neurons, and degeneration of the substantia nigra have been emphasized in previous descriptions. The minimal pathologic features required for a diagnosis of CBD are cortical and striatal tau-positive neuronal and glial lesions, especially APs and thread-like lesions in both white matter and gray matter; neuronal loss in focal cortical regions and in the substantia nigra are also prerequisites for a diagnosis of CBD (Table 1).

Reevaluation of the neuropathology in CBD cases Among the 30 pathologically confirmed CBD cases from our institute, 3 cases were associated with other neurologi-

cal diseases: one with motor neuron disease with TDP-43 proteinopathy, one with DLB, and one demonstrated a clinical history of cerebral palsy. The 27 cases that were analyzed had a mean age at onset of 63 years (range 51–79 years), a mean disease duration of 6 years (range 3–13 years), and a mean age at death of 69 years (range 54– 86 years). Although CBD is most often a sporadic disease, approximately 7% of affected individuals have a family history of neurological disorders, including PSP or dementia.

Macroscopic and microscopic findings Typical macroscopic findings (Fig. 4A) include hemiatrophy or bilateral asymmetrical cortical atrophy, which predominates in the perirolandic area, posterior-frontal to the parietal area, anterior frontal, or in the perisylvian area. Coronal sections demonstrate thinning of cortical ribbons associated with rarefaction of subcortical white matter in the affected cortices. Medial temporal structures are relatively preserved, but the brainstem and cerebellum may show variable atrophy. The substantia nigra consistently shows severe depigmentation, with variable atrophy of the basal ganglia including the globus pallidus and the subthalamic nucleus. The characteristic pathological hallmark of CBD is the swollen achromatic neuron, or ballooned cell (Table 1, Fig. 5a) that is present in affected cortical areas; however, ballooned neurons are nonspecific, and their quantity may vary according to the severity of the cortical involvement. Neuronal loss and gliosis are seen in the affected cortices, and typically, the degeneration of subcortical white matter is severe (Fig. 4Ac,d). Prominent cell loss and gliosis occur in the substantia nigra and are associated with varying degeneration of the globus pallidus and, to a lesser extent, of the subthalamic nucleus.The caudate and putamen show mild to moderate gliosis. The pathological hallmarks of CBD are hyperphosphorylated 4R tau- positive neuronal and glial inclusions, pretangles, NFTs, threads, APs, and coiled bodies (Table 1, Fig. 5). Immunohistochemistry for the detection of hyperphosphorylated tau reveals these abnormal tau aggregates, but they are most conspicuous after GB silver staining.

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Fig. 4 Macroscopic findings in typical CBD (A) and PSP-like CBD (B). (A) Macroscopic findings in typical CBD show right sidepredominant bilateral cerebral atrophy (a). A coronal section demonstrates right side-predominant frontal atrophy, thinning of cortical ribbons and subcortical white matter, and atrophy of the globus pallidus and subthalamic nucleus associated with atrophy of the putamen and caudate (b). Medial temporal structures are relatively preserved. The same specimens as in panel (b) demonstrate right sidepredominant atrophy of cortical and basal ganglia with reduced Klüver-Barrera staining of subcortical white matter including U-fibers (c), and this is shown by Holzer staining to be accompanied with fibrous gliosis (d). Bar = 2 cm. (B) Macroscopic findings in PSP-like CBD indicate severe atrophy of the pallidum and subthalamic nucleus with relatively mild degeneration of the cerebral cortices and subcortical white matter (a, b) and severe depigmentation of the substantia nigra accompanied by atrophy of the brainstem tegmentum (c, d) and dentate nucleus (e). Microscopically, astrocytic plaques are found in the basal ganglia and cerebral cortices (f). In the pontine nucleus, numerous pretangles, threads and neurites are found (g). b, d, e, Klüver-Barrera staining and Holzer stain. a, c, bar = 2 cm; f, bar = 10 μm; g, bar = 50 μm.

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Fig. 5 Major microscopic findings in CBD. Microscopic findings in CBD include ballooned neurons (a), numerous tau-positive threads in cortical and subcortical structures (b), pretangles (c) and APs (d). An AP has Gallyas-positive (e) and 4R tau-positive (f) filamentous structures associated with collaterals (g) in a coronal arrangement. A double-immunofluorescence study of APs indicates a tau-positive coronal arrangement of filaments (h, red) located into the distal portion of GFAP-positive astrocytic processes (green), which are shown merged only within the distal portions (i). a, H&E; b, c, d, g, AT8; e, Gallyas silver stain; f, RD4 immunohistochemistry; h, i, doubleimmunofluorescence for tau (red) and GFAP (green). a, d, g, bar = 10 μm; b, bar = 50 μm; c, f, bar = 20 μm. e, bar = 100 μm.

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Fig. 6 Ultrastructural characterization of AP. Lower magnification of an AP, indicate Q dot-labeled AT8-positive short filaments (black arrow) loosely bundled near the synaptic structures and glial filaments (white arrow) (a). Higher magnification (b) indicates Q dot-labeled tau-positive short filamentous structures (black arrow) are inserted into a narrow gap near the synaptic structures. These short filamentous structures may represent the collateral structures of APs.

Although threads, pretangles and APs are highly specific for CBD, APs are almost exclusively seen in CBD and are considered a diagnostic hallmark.

Definition and differentials of astrocytic plaques APs are not visualized in hematoxylin-eosin staining, but GB silver staining and tau immunohistochemistry reveal their distinctive morphology. APs exhibit a corona-like arrangement and are composed of fuzzy, short processes with tapered ends and fine collaterals, which were more evident on GB silver staining (Fig. 5d–g). No cytoplasmic staining was observed within the APs.6 APs were first identified by Feany and Dickson 37 using immunohistochemistry and laser confocal microscopy. They demonstrated that the nonamyloid cortical plaques of CBD are actually collections of abnormal tau in the distal processes of astrocytes, which were defined as APs. These glial cells express both vimentin and CD44, markers of astrocyte activation. Differential structures are threads, TAs, other taupositive astrocytic inclusions and senile plaques. Threads are fine, thin filamentous structures that are abundant in the cerebral cortices, subcortical white matter, basal ganglia, and brain stem; they are not corona-like in appearance and do not have collaterals or a bush-like appearance. Senile plaques are composed of amyloid β accompanied by tau-positive dystrophic neurites. TAs in PSP have been described previously. Using double immunofluorescence staining, APs were found to be partially demonstrated colocalization with GFAP and tau in the distal portion of GFAP- positive asrtrocytic processes (Fig. 5h,i). Colocalization of GFAP and tau was not clearly observed in the cytoplasm or in the proximal processes of astrocytes. Immunoelectron micro© 2014 Japanese Society of Neuropathology

scopic examination was performed using the preembedding Q-dots immunolabeling, which was modified from the previously reported method.38–40 It revealed that an AP contained a randomly arranged bundle of straight and twisted tubules in 15–20 nm diameter and the taupositive filaments were densely immunolabeled with Q-dots (Fig. 6). Some of these filaments are located in a narrow hollow near synaptic structures (Fig. 6b). These findings suggested that the bush-like or collateral appearance of tau-positive APs may reflect the collaterals of distal astrocytic processes, which serve as a cover layer for the dendritic and perikaryal surfaces of certain neurons, and groups of, or individual synapses.41 The location of filamentous tau accumulation of APs in the immediate vicinity of the synaptic structures suggest that APs may impair synaptic functions. APs were most abundant in the prefrontal and premotor areas in the cerebral cortex of brains from individuals diagnosed with CBD, but many APs were also observed in the caudate nucleus.42 They are rarely seen in white matter with numerous tau-positive threads, despite severe gliosis. This also suggests that the morphology of the processes of protoplasmic astrocytes in the gray matter may be closely related to the corona-like structures of APs (Figs 7,8). The origin of tau-positive filaments in APs is still unknown. The density of APs is variable according to the degree and extent of the disease of the affected cortices. Even when the degeneration of the cerebral cortices is mild, a small number of typical APs are enough for a diagnosis of CBD.

Correlation between APs and clinicopathological features Based on the distribution of neuronal loss with gliosis and tau positive neuronal and glial inclusions in the 27 CBD

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Fig. 7 Schematic illustration of tau accumulation in TAs of PSP and APs of CBD. In TAs (a), radially arranged filamentous tau aggregation (red lines) occurs in the cytoplasm and proximal processes of astrocytes. In APs (b), annularly distributed tau aggregation (green lines) is formed in the most distal portion of the astrocytic processes associated with collaterals. (Modified diagram reproduced with the permission of Igaku-Shoin from “A Guide to Neuropathology” by Hirano A, p205, Tokyo, 1981)40

cases, pathological topography was grossly divided into 3 representative subtypes: typical CBD type, basal ganglia predominant type, and PSP-like type (Fig. 1B). Typical CBD type (15 out of 27 cases, 56%) represents prototypical CBD and involves asymmetrical degeneration of the cerebral cortices and subcortical white matter, substantia nigra, pallidum, and the subthalamic nucleus, from a moderate to a severe extent (Fig. 1B). In the typical CBD type, the cortical distribution was predominantly in the posterior frontoparietal and perisylvian areas in 10 cases (37%), and in the anterior frontal area in 5 cases (19%). The PSP-like type (9 cases, 33%) revealed prominent involvement of the basal ganglia, brainstem and cerebellar dentate nucleus with mild to moderate involvement of the cerebral cortices without obvious laterality (Fig. 1B,4B). The basal gangliapredominant type (3 cases, 11%) demonstrated relatively confined lesions to the pallidum, subthalamic nucleus, and the substantia nigra, with mild degeneration of the cerebral cortices, white matter, brainstem and cerebellum (Fig. 1B).43 The pathological subtypes wholly correspond to the clinical phenotypes: pathologically typical CBD type with posterior frontoparietal or perisylvian cortical lesions corresponds to CBD with clinical CBS; similarly, the pathological PSP-like type corresponds to CBD with clinical PSP syndrome. The pathologically typical CBD type with ante-

rior frontal involvement corresponds to the clinical phenotype FBS, and the basal ganglia-predominant type demonstrates atypical parkinsonism or PSPS. Therefore, clinical phenotypes depend on the topographical distribution and degree of degeneration of the cerebral cortices, basal ganglia, and brainstem. In fact, we analyzed cases that exhibited borderline pathology between those pathological subtypes. APs are exclusively seen in all CBD subtypes, although the density is variable according to the cerebral regions that are affected. APs are usually prominent in the frontoparietal cortices and in the striatum, especially in the caudate.43 In the typical CBD type, more prominent APs are recognized in the affected cerebral cortices and in the striatum. In the PSP-like type or the basal ganglia- predominant type, APs are located in the putamen and caudate and to a lesser extent in the cerebral cortices. APs typically associate with pretangles and threads.

UNDERLYING MECHANISM Although PSP and CBD are independent 4R tauopathies, highly overlapping pathological distributions seem to influence clinical diagnostic practices. The microscopic tau pathology allows for the clear discrimination of distinctive tau aggregates in neurons and glia between the two © 2014 Japanese Society of Neuropathology

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Fig. 8 Schematic illustration of tau accumulation in neurons and glia in PSP and CBD. In PSP (left panel), filamentous tau aggregation (red lines) is produced in the form of globose-type NFTs, TAs, and coiled bodies within the soma and proximal processes of neurons and glia. In CBD (right panel) less fibrillar tau aggregation (yellow lines) is produced and thus the majority of aggregates appear in the form of pretangles, APs, and threads within the cytoplasm of the soma and the distal processes of neurons and glia.

diseases. In this review, colocalization of TAs and APs was not found, although colocalization of TAs and APs as well as the coexistence of PSP and CBD in the same family were reported in some cases.44–46 In the TAs of PSP, tau accumulates in the proximal portion of astrocytic processes and the perikaryon, in contrast to tau accumulation in the distal processes of astrocytes in the APs of CBD (Fig. 7). Tau accumulation of APs in the immediate vicinity of the synaptic structures suggests synaptic dysfunction by APs, which may provide the different pathophysiological mechanism from that of AD with predominant neuronal tau accumulation. Immunoelectron microscopic examination In Furthermore, in PSP, neuronal tau aggregates form a fibrillar, globose type of NFT, while in CBD, less filamentous tau accumulates in neurons as pretangles (Fig. 8).47,48 On immunoblots of sarkosyl-insoluble brain extracts, a 33-kDa band predominated in the low molecular weight tau fragments in PSP, whereas two closely related bands of approximately 37 kDa predominated in CBD. These results suggest that despite the identical composition of the © 2014 Japanese Society of Neuropathology

tau isoforms, distinct proteolytic processing of abnormal tau occurs in these two diseases.49

CONCLUSIONS Astrocytic inclusions in PSP and CBD are briefly reviewed along with the pathological characteristics of tau aggregation and the pathological diversity of PSP and CBD. The different sites of tau aggregation in astrocytes and the different extent of fibril formation in neurons might help differentiate these two 4R tauopathies (Fig. 8).

Addendum Dr. Nakano I. from Tokyo Metropolitan Neurological Hospital questioned the certainty of immunoelectron microscopic findings and the relationship between the APs and vessels. The double immunohistochemistry analysis of vimentin and AT8 in the frontal cortices of CBD patients indicated that the processes of astrocytes within APs rarely

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Fig. 9 Perivascular orientation of APs. Double immunohistochemistry for vimentin (violet) and AT8 (brown) of the frontal cortices in CBD indicates that the astrocytic processes radiate continuously to AT8-positive APs (white arrow) and rarely contact vessels (V), although some of the processes attached to the vessels showed AT8-positive staining (black arrow). V, vessel; A, astrocyte; bar = 10 μm.

contact vessels. Tau deposition into the perivascular foot processes was not remarkable, although some of the processes attached to the vessels demonstrated AT8-positive staining (Fig. 9).50

COI The author declares no competing interests.

ACKNOWLEDGMENTS This work was presented in part at the 54th Annual Meeting of the Japanese Society of Neuropathology (Tokyo, Japan, 2013) and was supported by grants from the Collaborative Research Project [2012-2308 to MY] of the Brain Research Institute, Niigata University and grants-in-aid from the Research Committee of CNS Degenerative Diseases, the Ministry of Health, Labour and Welfare of Japan. The author acknowledges Dr. S. Tatsumi, Dr. M. Mimuro, and Dr. Y. Iwasaki, and also thanks K. Koutani, T. Mizuno, C. Sano and C. Uno for excellent technical assistance.

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