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P301L tauopathy
Journal of the Neurological Sciences 200 (2002) 85 – 93 www.elsevier.com/locate/jns
P301L tauopathy: confocal immunofluorescence study of perinuclear aggregation of the mutated protein Emil Adamec a,*, Jill R. Murrell b, Masaki Takao b, Wendy Hobbs c, Ralph A. Nixon d,e, Bernardino Ghetti b, Jean P. Vonsattel f a
Department of Psychiatry, Harvard Medical School, Mailman Research Center, Laboratories for Molecular Neuroscience, McLean Hospital, 115 Mill Street, Belmont, MA 02478, USA b Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA c Department of Pathology, Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114, USA d Department of Psychiatry, New York University School of Medicine and Nathan Kline Institute, Orangeburg, NY 10962, USA e Department of Cell Biology, New York University School of Medicine and Nathan Kline Institute, Orangeburg, NY 10962, USA f Department of Pathology, Columbia University, Columbia Presbyterian Medical Center, New York, NY 10032, USA Received 4 February 2002; received in revised form 18 April 2002; accepted 13 May 2002
Abstract The clinical and neuropathological features in the P301L tauopathy have been described in several kindreds. In this study, we present findings in two previously unreported patients, evaluated both genetically, neuropathologically, and with multiparametric confocal immunofluorescence. The patients were female, with age 65 and 75 years old, respectively. Both exhibited clinical symptoms of frontotemporal dementia (FTD). Marked atrophy of the frontal and temporal lobes with moderate atrophy of the remaining cerebral and brain stem structures was present. The substantia nigra was pale. The atrophic neocortical regions exhibited neuronal loss, marked gliosis, status spongiosus, and occasional ballooned neurons. By light microscopy, the most striking findings were argyrophilic perinuclear rings, frequently with an attached small inclusion (mini Pick-like body), especially prominent in dentate granule cells, entorhinal and temporal cortices, and to a lesser extent in CA1. These structures were immunopositive for tau protein (Tau-2, AT-8, PHF-1, MC-1). Numerous astrocytic plaques, tuft-shaped astrocytes, coiled bodies, and dystrophic neurites were also present. Confocal immunofluorescence with a P301L-specific antibody directly demonstrated the presence of the mutated protein in the PHF-1 positive aggregates. The mutated tau protein (4-repeat tau) was detected in the mini Pick-like bodies, indicating an important biochemical difference between these inclusions and classical Pick bodies (3-repeat tau). Additionally, since 4-repeat tau protein is not normally present in dentate granule cells, this result also suggests an abnormality in the mRNA splicing mechanisms. The structural features of the involvement of proteolytic systems in this tauopathy were assessed by immunohistochemistry for the active form of calpain II (C-27) and ubiquitin. Colocalization of PHF-1 positive aggregates with C-27 points to the possible involvement of calpain in tau protein hyperphosphorylation. Absence of immunostaining for ubiquitin indicates possible dysfunction of the ubiquitin – proteasome system in this tauopathy. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Tau protein; Tauopathy; Mutation; Frontotemporal dementia; Immunohistochemistry; Confocal microscopy
1. Introduction The syndrome of frontotemporal dementia (FTD) is a clinical term relating to a non-Alzheimer type of dementia characterized by personality changes, prominent loss of executive function, and deterioration of memory [1 – 3].
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Corresponding author. Tel.: +1-617-855-3804; fax: +1-617-855-3479. E-mail address: [email protected] (E. Adamec).
Later on, stereotypical behavior and apathy also become prominent. Some patients exhibit also parkinsonian symptomatology. A subset of hereditary cases of FTD with a link to chromosome 17q21 – 22 [4] is now called ‘‘frontotemporal dementia and parkinsonism linked to chromosome 17’’ (FTDP-17, [3,5]). FTDP-17 patients show autosomal dominant mode of inheritance with age-dependent penetrance. Most cases have an age of onset 45 – 65 years [5– 7]. Genetically, FTDP-17 cases are characterized by a mutation (missense, deletion, or splice site) in the gene for the
0022-510X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 5 1 0 X ( 0 2 ) 0 0 1 5 0 - 8
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microtubule-associated protein tau [8– 11]. In adult human brain, mRNA from a single tau gene [12,13] undergoes alternative splicing, leading to the presence of six tau protein isoforms. They differ by the presence or absence of two inserts in the N-terminal half and one insert in the Cterminal half of the protein. Isoforms containing the insert in the C-terminal half (encoded by exon 10) are known as 4repeat isoforms. Isoforms lacking the insert are called 3repeat isoforms. The repeats constitute the microtubulebinding domains of tau [11,14,15]. Neuropathologically, FTDP-17 brains exhibit prominent atrophy of the frontal and temporal lobes, basal ganglia, and substantia nigra, with an accompanying neuronal loss and gliosis. Argyrophilic cytoplasmic inclusions containing tau protein are present in many neurons and, in some instances, also in the glia [3]. The biochemistry and the pathophysiology of various tau mutations have been recently reviewed [7,16]. The P301L mutation is the most frequently encountered tauopathy [17,18], making the pathological features of this FTDP-17 of considerable importance to neuropathologists. In this study, we present two previously unreported patients with findings of interest, both in the differential diagnosis of FTD and in the elucidation of the molecular pathophysiology of this tauopathy. Part of this study has been presented in abstract form [19].
2. Materials and methods 2.1. Neuropathology Brains were collected for diagnosis and research at the Harvard Brain Tissue Resource Center at McLean Hospital (Belmont, MA) according to a previously described protocol [20]. Briefly, one-half brain was immersion fixed in 10% buffered formalin solution, 18 representative blocks were taken, were embedded in paraffin, and were processed for neuropathological evaluation. Seven micrometer sections from all blocks were stained with hematoxylin and eosin/ Luxol-fast-blue, Bielschowsky silver impregnation, selected sections were stained with Congo red. Representative blocks from the contralateral half brain of b3630 were frozen in either liquid nitrogen vapor or on dry ice. Only formalin fixed tissue was available from brain b4097. 2.2. Immunohistochemistry and confocal microscopy Deparaffinized and rehydrated sections were exposed to primary antibodies by overnight incubation at 4 jC. For
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Fig. 2. Direct sequencing of exon 10 of the tau gene from the proband (b3630 shown) and a normal control. This shows the sequence of the negative strand. A C-to-T transition (arrow) in codon 301 results in a proline to leucine amino acid change in the tau protein.
light microscopy, the detection was performed with either the avidin –biotin – peroxidase technique (ABC, Vector, Burlinghame, CA) and diaminobenzidine as chromogen or with the biotin –streptavidin alkaline phosphatase technique with fuchsin as chromogen. For the antibodies Ab P301L and C27, the antigen retrieval technique was used according to the manufacturer’s instructions (Vector). Labeling for immunofluorescence was done with isotype-specific secondary antibodies tagged with Cy2 or Cy5 ([21], Jackson ImmunoResearch, West Grove, PA). Nuclei were visualized with the DNA dye Sytox orange ([22], Molecular Probes, Eugene, OR). Confocal imaging was performed on a Leica TCS NT system connected to an inverted microscope [23]. 2.3. Antibodies The following, previously characterized, primary antibodies were used: mouse monoclonal phosphorylation-independent antibody tau-2 (1:7500, Sigma, St. Louis, MO), mouse monoclonal AT-8 (1:200, recognizes tau phosphorylated at both serine 202 and threonine 205 [24,25], Innogenetics, Norcross, GA), mouse monoclonal PHF-1 (1:250, recognizes tau protein phosphorylated at both serine 396 and serine 404 [26,27], P. Davies), mouse monoclonal MC1 (recognizes a conformational epitope on tau protein [28], P. Davies), rabbit polyclonal anti-ubiquitin (recognizes primarily poly-ubiquitinated substrates,1:300, Dako, Carpenteria, CA), mouse monoclonal anti-GFAP (1:240, Dako),
Fig. 1. Immunohistochemistry on sections from the entorhinal cortex shows neuronal loss, prominent gliosis, microvacuolization of layer II, and absence of amyloid plaques or Lewy bodies (A – C). On Bielschowsky preparations, perinuclear rings, some with a small round inclusion (mini Pick-like body), were especially prominent in dentate granule cells (D, E) and CA1 (F). Immunostaining on sections from the dentate gyrus (G – I), the entorhinal (J) and temporal cortices (K) demonstrated that these structures contained tau protein. Tau-positive, tuft-shaped astrocytes in the molecular layer of the dentate gyrus are shown in L. Bar in A – C, I, K, and L: 100 Am; in D, G, H, and J: 50 Am; in E and F: 10 Am.
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mouse monoclonal anti-Ah (1:150, Dako), mouse monoclonal anti-a-synuclein (H3C, [29], J. George), rabbit polyclonal antibody which specifically recognizes tau protein with the P301L mutation (Ab P301L, [30], P. Rizzu and P. Heutink), and rabbit polyclonal antibody C-27 (recognizes the active form of calpain II, [31]). 2.4. Genetic analysis Genomic DNA was extracted either from frozen cerebellum (b3630) or from paraffin-embedded tissue (b4097). PCR amplification and sequencing was done as described in detail previously [32].
3. Results Both brains were evaluated as part of a brain donation program at the Harvard Brain Tissue Resource Center at McLean Hospital. Only a limited amount of information on family history and the course of disease was available. The first patient (b3630) was a female of North European ancestry. She presented at 53 years old with attention deficit, apathy, placidity, hyperorality, hyperphagia, and lack of insight. Initially, her memory was relatively well preserved. The disease progression was characterized by memory worsening and she died at age 65. Her mother began showing signs of dementia in her early 50s and died profoundly demented at age 72. The second patient (b4097) was a female of French Canadian ancestry who presented at age 70 with aphasia and mild dementia and died at age 75. Fresh brain weight was 925 g for b3630 and 1100 g for b4097. Both brains exhibited quite similar neuropathological findings. Macroscopically, frontal and temporal lobes were prominently atrophic, moderate atrophy was present in the remaining cerebral and brain stem structures. Substantia nigra was pale. Locus coeruleus of b3630 was well pigmented, but pale in b4097. By light microscopy, the atrophic regions showed severe neuronal loss, scattered ballooned neurons, status spongiosus (esp. upper cortical layers), and marked gliosis, prominent in superficial cortical layers and in the cortico-subcortical junction (Fig. 1A). Rare diffuse plaques and rare Pick bodies were detected in b3630. Mature neuritic plaques of Alzheimer and Lewy bodies were absent in both brains (Fig. 1B,C). A striking finding in Bielschowsky preparations were many neurons with perinuclear argyrophilic rings, in many instances with an attached small round body (Fig. 1D – F),
especially frequent in temporal and entorhinal cortices, dentate granule cells, and to a lesser extent, in the CA1 region of the hippocampus. These structures were immunopositive with anti-tau antibodies, including those that were phosphorylation-independent (tau-2), phosphorylationdependent (AT-8 and PHF-1), or conformation-dependent (MC-1) (Fig. 1G – L). Additionally, anti-tau antibodies labeled numerous astrocytic plaques, tuft-shaped astrocytes (Fig. 1L), oligodendroglial coiled bodies, and neuronal processes. Tau-immunopositivity was strong in the neocortex, amygdala, dentate gyrus, substantia innominata, substantia nigra, oculomotor nucleus, periaqueductal gray, raphe nuclei, and locus coeruleus. Direct sequencing of PCR amplified exons of the tau protein gene identified in both patients a heterozygous P301L mutation (CCG to CTG in exon 10 of tau gene, Fig. 2). A novel antibody specifically recognizing tau protein with the P301L amino acid substitution (Ab P301L) was used to determine if the aggregates of phosphorylated tau protein contain also the mutated protein [30]. On brightfield preparations (Fig. 3A – C), the staining pattern with the Ab P301L was quite similar to that observed with standard antitau antibodies (Fig. 1G – L). Multiparametric confocal microscopy utilizing double labeling with Ab P301L and PHF-1 revealed that essentially all PHF-1 positive perinuclear rings, small attached inclusions, and larger neuritic processes do indeed contain the mutated tau protein (Fig. 3D –L). The mutated protein was also present in the short thread-like structures in the neuropil, even though the PHF1 labeled threads were slightly more numerous. In both patients, immunohistochemistry for ubiquitin (Fig. 4A – C) showed a complete absence of labeling of the tau-positive rings, small inclusions, and neuritic processes, even though a rare tangle of Alzheimer in the entorhinal cortex was clearly labeled (Fig. 4A). This finding points to a possible role of abnormalities in proteolytic mechanisms as a putative mechanism of neurodegeneration in this tauopathy. Previous studies have established that neurofibrillary tangles and neuropil threads in Alzheimer’s disease label with active site-directed antibodies to calpain II (C-18 and C-27, [31]), one of the isoforms of a family of calcium-dependent cysteine proteases, an important proteolytic system involved in a variety of cellular functions. With light microscopy, the antibody C-27 labeled neurofibrillary tangles and dystrophic neurites in preparations from Down’s syndrome brain (Fig. 4D, male 42 years). In P301L tauopathy, the perinuclear rings, dystrophic processes, and rare Pick bodies were also labeled (Fig. 4E,F). In both P301L
Fig. 3. Immunohistochemistry with an antibody specifically recognizing tau protein with the P301L mutation (Ab P301L) evaluated in light microscopy demonstrates the presence of the mutated protein in many perinuclear rings in dentate granule cells (A), pyramidal neurons in parahippocampal gyrus (B), and Alzheimer-type tangles in CA2 (C). The colocalization (appears yellow in overlay images) of the mutated (Ab P301L, red) and hyperphosphorylated (PHF-1, green) tau protein in larger aggregates and dystrophic neurites was directly demonstrated by triple-label confocal immunofluorescence (D – L). Smaller neuropil threads were positive mostly for PHF-1. The images were acquired with a Leica TCS NT confocal system and represent single sections through the middle of the preparation. D – F are from dentate granule cells, G – I are from pyramidal neurons in the parahippocampal gyrus, and J – L are from CA1. In F, I, and L, the merged green and red images were overlaid with blue representing nuclear staining. Bar in A – C: 50 Am; in D – L: 25 Am.
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Fig. 4. Immunohistochemistry for ubiquitin (A – C) demonstrates absence of labeling of the tau protein aggregates in the entorhinal cortex (A) and dentate granule cells (B – C) even though a rare tangle of Alzheimer is clearly labeled (A). Antibody to the active form of calpain II (C-27) labels tangles and dystrophic neurites in Down’s syndrome (D, positive control for C-27 immunohistochemistry). In P301L tauopathy, the active form of calpain II colocalizes with PHF-1 positive perinuclear aggregates and some dystrophic neurites, both in light (E – F) and confocal (G – I) microscopy.Bar in A – F: 50 Am; in G – I: 25 Am.
brains, the evaluation by double-label confocal immunofluorescence with PHF-1 and C-27 (Fig. 4G – I) directly demonstrated colocalization of the active form of calpain II in the PHF-1 positive aggregates.
4. Discussion Extensive literature exists on clinical, pathological, and biochemical findings in the P301L tauopathy [17,18,30,32–
37]. The overall pathological features of the two brains studied in this report were consistent with previous descriptions. There were, however, several features of considerable interest, relating to both the differential diagnosis and the pathogenesis of this disorder. To further extend our knowledge about the pathophysiology involved, in addition to standard neuropathological evaluation of tauopathies, we have focused on two important issues. First, we have addressed the question of whether the aggregates of hyperphoshorylated tau do indeed contain
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the mutated protein. Both brains exhibited a striking perinuclear aggregation of argyrophilic, hyperphosphorylated tau proteins in the form of a ring, frequently with an attached small inclusion. These were especially prominent in dentate granule cells, and to a lesser extent in CA1, entorhinal, and temporal cortices. Utilizing a novel antibody specifically recognizing tau protein with the P301L mutation, we have directly demonstrated that both the rings and the attached inclusions contain the mutated protein. The finding of the mutated protein in dentate granule cells is of special significance. First, in FTD cases with prominent frontotemporal atrophy, the evaluation of dentate granule cells focuses on establishing the presence or absence of inclusions, Pick bodies being the most common. The small inclusion described here could be called a mini Pick-like body and is possibly considered to represent an early stage of the formation of a classical Pick body. However, since Pick bodies contain only 3-repeat tau isoforms [38,39], the presence of tau protein with the P301L mutation (4-repeat tau) clearly points to important biochemical differences between these types of inclusion bodies, a finding of direct relevance in the differential diagnosis of Pick’s disease. Second, since dentate granule cells normally express only 3-repeat tau isoforms [40], the presence of the mutated protein indicates an abnormality in mRNA splicing. This finding is of potentially high importance in the understanding of the pathophysiology of tauopathies in general, since it has been recently described also in the dentate fascia in Alzheimer’s disease and progressive supranuclear palsy [41]. The second important issue addressed in this report was the morphological assessment of proteolytic systems in P301L tauopathy. Abnormalities in proteolysis are being considered as one of the putative mechanisms of neurodegeneration in tauopathies [16]. Recent studies indicate that the calcium-dependent cysteine protease calpain [42] is involved in several important ways in tau protein hyperphosphorylation and neurodegeneration in general [43 – 45]. The regulatory subunit p35 of cyclin-dependent kinase 5 (Cdk5), one of the kinases involved in tau phosphorylation, is cleaved by calpain to p25. Binding of p25 to Cdk5 constitutively activates Cdk5, changes its cellular location, and alters its substrate specificity. Based on these studies, it is quite possible that the association of the active form of calpain II (C-27) with PHF-1 labeling reflects Cdk5 activation, one of the protein kinases implicated in tau hyperphosphorylation. An alternative explanation for the C-27/ PHF-1 colocalization would be the involvement of calpain in the proteolysis of tau protein. Tau incorporated into paired helical filaments (PHFs) is considerably more resistant to proteolysis by calpain [46], which could explain both the persistence of these structures and also the prominent calpain activation as a persistent attempt to degrade the abnormal protein. A recent report [47] also indicates that, at least in biochemical studies, tau proteins with the mutations V337M or R406W are less susceptible than either tau with the P301L mutation or corresponding wild-type tau to
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degradation by calpain I, the other main isoform of the protease. The differences were, at least in part, due to changes in accessibility of a cleavage site located about 100 amino acids from the C-terminus. Even though the exact interpretations of these findings will depend on further biochemical studies and the availability of antibodies, these findings nevertheless point to the important role of the calpain system in the pathophysiology of this tauopathy. The ubiquitin– proteasome complex represents another proteolytic system involved in the degradation of a variety of cellular proteins [48 – 50]. The complete lack of immunostaining of the tau aggregates for ubiquitin points to a possible abnormality in this system in the P301L tauopathy. One mechanism might involve inefficient attachment of ubiquitin by conjugating enzymes. Alternatively, tau protein might be properly conjugated, targeted to proteasome, deubiquitinated, but inefficiently degraded with subsequent aggregation in the proteasome complex. Finally, incomplete penetration of the anti-ubiquitin antibody needs to be considered. Whether the tau-positive perinuclear rings do indeed represent accumulation of undegraded tau in the proteasome awaits further ultrastructural studies. The findings nevertheless indicate that further studies need to elucidate the molecular mechanisms of proteolysis in tauopathies since they might represent important pathophysiological mechanisms of neurodegeneration in this disorder. Acknowledgements We would like to thank Dr. A. Andreadis (Eunice Kennedy Shriver Center, Waltham, MA) for helpful advice. We would also like to thank Ms. Karlotta Fitch and Mr. Timothy Wheelock for help with the preparation of microscopic slides. The antibodies PHF-1 and MC-1 were kindly provided by Dr. P. Davies (Albert Einstein College of Medicine, Bronx, NY), the antibody H3C by Dr. J.M. George (University of Illinois, Urbana-Champain, IL). Drs. P. Rizzu and P. Heutink (Erasmus University, Rotterdam, The Netherlands) kindly provided the antibody P301L. This work was supported by grants from the National Institute on Aging, AG00764, AG05134, AG17617, and AG10133. The Harvard Brain Tissue Resource Center is supported in part by PHS grant MH/NS 31862. References [1] Gustafson L. Frontal lobe degeneration of non-Alzheimer type: II. Clinical picture and differential diagnosis. Arch Gerontol Geriatr 1987;6:209 – 23. [2] Neary D, Snowden JS, Northen B, Goulding P. Dementia of frontal lobe type. J Neurol Neurosurg Psychiatry 1988;51:353 – 61. [3] Spillantini MG, Bird TD, Ghetti B. Frontotemporal dementia and Parkinsonism linked to chromosome 17: a new group of tauopathies. Brain Pathol 1998;8:387 – 402. [4] Wilhelmsen KC, Lynch T, Pavlou E, Higgins M, Nygaard TG. Localization of disinhibition – dementia – parkinsonism – amyotrophy complex to 17q21 – 22. Am J Hum Genet 1994;55:1159 – 65.
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P301L tauopathy: confocal immunofluorescence study of perinuclear aggregation of the mutated protein Emil Adamec a,*, Jill R. Murrell b, Masaki Takao b, Wendy Hobbs c, Ralph A. Nixon d,e, Bernardino Ghetti b, Jean P. Vonsattel f a
Department of Psychiatry, Harvard Medical School, Mailman Research Center, Laboratories for Molecular Neuroscience, McLean Hospital, 115 Mill Street, Belmont, MA 02478, USA b Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA c Department of Pathology, Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114, USA d Department of Psychiatry, New York University School of Medicine and Nathan Kline Institute, Orangeburg, NY 10962, USA e Department of Cell Biology, New York University School of Medicine and Nathan Kline Institute, Orangeburg, NY 10962, USA f Department of Pathology, Columbia University, Columbia Presbyterian Medical Center, New York, NY 10032, USA Received 4 February 2002; received in revised form 18 April 2002; accepted 13 May 2002
Abstract The clinical and neuropathological features in the P301L tauopathy have been described in several kindreds. In this study, we present findings in two previously unreported patients, evaluated both genetically, neuropathologically, and with multiparametric confocal immunofluorescence. The patients were female, with age 65 and 75 years old, respectively. Both exhibited clinical symptoms of frontotemporal dementia (FTD). Marked atrophy of the frontal and temporal lobes with moderate atrophy of the remaining cerebral and brain stem structures was present. The substantia nigra was pale. The atrophic neocortical regions exhibited neuronal loss, marked gliosis, status spongiosus, and occasional ballooned neurons. By light microscopy, the most striking findings were argyrophilic perinuclear rings, frequently with an attached small inclusion (mini Pick-like body), especially prominent in dentate granule cells, entorhinal and temporal cortices, and to a lesser extent in CA1. These structures were immunopositive for tau protein (Tau-2, AT-8, PHF-1, MC-1). Numerous astrocytic plaques, tuft-shaped astrocytes, coiled bodies, and dystrophic neurites were also present. Confocal immunofluorescence with a P301L-specific antibody directly demonstrated the presence of the mutated protein in the PHF-1 positive aggregates. The mutated tau protein (4-repeat tau) was detected in the mini Pick-like bodies, indicating an important biochemical difference between these inclusions and classical Pick bodies (3-repeat tau). Additionally, since 4-repeat tau protein is not normally present in dentate granule cells, this result also suggests an abnormality in the mRNA splicing mechanisms. The structural features of the involvement of proteolytic systems in this tauopathy were assessed by immunohistochemistry for the active form of calpain II (C-27) and ubiquitin. Colocalization of PHF-1 positive aggregates with C-27 points to the possible involvement of calpain in tau protein hyperphosphorylation. Absence of immunostaining for ubiquitin indicates possible dysfunction of the ubiquitin – proteasome system in this tauopathy. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Tau protein; Tauopathy; Mutation; Frontotemporal dementia; Immunohistochemistry; Confocal microscopy
1. Introduction The syndrome of frontotemporal dementia (FTD) is a clinical term relating to a non-Alzheimer type of dementia characterized by personality changes, prominent loss of executive function, and deterioration of memory [1 – 3].
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Corresponding author. Tel.: +1-617-855-3804; fax: +1-617-855-3479. E-mail address: [email protected] (E. Adamec).
Later on, stereotypical behavior and apathy also become prominent. Some patients exhibit also parkinsonian symptomatology. A subset of hereditary cases of FTD with a link to chromosome 17q21 – 22 [4] is now called ‘‘frontotemporal dementia and parkinsonism linked to chromosome 17’’ (FTDP-17, [3,5]). FTDP-17 patients show autosomal dominant mode of inheritance with age-dependent penetrance. Most cases have an age of onset 45 – 65 years [5– 7]. Genetically, FTDP-17 cases are characterized by a mutation (missense, deletion, or splice site) in the gene for the
0022-510X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 5 1 0 X ( 0 2 ) 0 0 1 5 0 - 8
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microtubule-associated protein tau [8– 11]. In adult human brain, mRNA from a single tau gene [12,13] undergoes alternative splicing, leading to the presence of six tau protein isoforms. They differ by the presence or absence of two inserts in the N-terminal half and one insert in the Cterminal half of the protein. Isoforms containing the insert in the C-terminal half (encoded by exon 10) are known as 4repeat isoforms. Isoforms lacking the insert are called 3repeat isoforms. The repeats constitute the microtubulebinding domains of tau [11,14,15]. Neuropathologically, FTDP-17 brains exhibit prominent atrophy of the frontal and temporal lobes, basal ganglia, and substantia nigra, with an accompanying neuronal loss and gliosis. Argyrophilic cytoplasmic inclusions containing tau protein are present in many neurons and, in some instances, also in the glia [3]. The biochemistry and the pathophysiology of various tau mutations have been recently reviewed [7,16]. The P301L mutation is the most frequently encountered tauopathy [17,18], making the pathological features of this FTDP-17 of considerable importance to neuropathologists. In this study, we present two previously unreported patients with findings of interest, both in the differential diagnosis of FTD and in the elucidation of the molecular pathophysiology of this tauopathy. Part of this study has been presented in abstract form [19].
2. Materials and methods 2.1. Neuropathology Brains were collected for diagnosis and research at the Harvard Brain Tissue Resource Center at McLean Hospital (Belmont, MA) according to a previously described protocol [20]. Briefly, one-half brain was immersion fixed in 10% buffered formalin solution, 18 representative blocks were taken, were embedded in paraffin, and were processed for neuropathological evaluation. Seven micrometer sections from all blocks were stained with hematoxylin and eosin/ Luxol-fast-blue, Bielschowsky silver impregnation, selected sections were stained with Congo red. Representative blocks from the contralateral half brain of b3630 were frozen in either liquid nitrogen vapor or on dry ice. Only formalin fixed tissue was available from brain b4097. 2.2. Immunohistochemistry and confocal microscopy Deparaffinized and rehydrated sections were exposed to primary antibodies by overnight incubation at 4 jC. For
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Fig. 2. Direct sequencing of exon 10 of the tau gene from the proband (b3630 shown) and a normal control. This shows the sequence of the negative strand. A C-to-T transition (arrow) in codon 301 results in a proline to leucine amino acid change in the tau protein.
light microscopy, the detection was performed with either the avidin –biotin – peroxidase technique (ABC, Vector, Burlinghame, CA) and diaminobenzidine as chromogen or with the biotin –streptavidin alkaline phosphatase technique with fuchsin as chromogen. For the antibodies Ab P301L and C27, the antigen retrieval technique was used according to the manufacturer’s instructions (Vector). Labeling for immunofluorescence was done with isotype-specific secondary antibodies tagged with Cy2 or Cy5 ([21], Jackson ImmunoResearch, West Grove, PA). Nuclei were visualized with the DNA dye Sytox orange ([22], Molecular Probes, Eugene, OR). Confocal imaging was performed on a Leica TCS NT system connected to an inverted microscope [23]. 2.3. Antibodies The following, previously characterized, primary antibodies were used: mouse monoclonal phosphorylation-independent antibody tau-2 (1:7500, Sigma, St. Louis, MO), mouse monoclonal AT-8 (1:200, recognizes tau phosphorylated at both serine 202 and threonine 205 [24,25], Innogenetics, Norcross, GA), mouse monoclonal PHF-1 (1:250, recognizes tau protein phosphorylated at both serine 396 and serine 404 [26,27], P. Davies), mouse monoclonal MC1 (recognizes a conformational epitope on tau protein [28], P. Davies), rabbit polyclonal anti-ubiquitin (recognizes primarily poly-ubiquitinated substrates,1:300, Dako, Carpenteria, CA), mouse monoclonal anti-GFAP (1:240, Dako),
Fig. 1. Immunohistochemistry on sections from the entorhinal cortex shows neuronal loss, prominent gliosis, microvacuolization of layer II, and absence of amyloid plaques or Lewy bodies (A – C). On Bielschowsky preparations, perinuclear rings, some with a small round inclusion (mini Pick-like body), were especially prominent in dentate granule cells (D, E) and CA1 (F). Immunostaining on sections from the dentate gyrus (G – I), the entorhinal (J) and temporal cortices (K) demonstrated that these structures contained tau protein. Tau-positive, tuft-shaped astrocytes in the molecular layer of the dentate gyrus are shown in L. Bar in A – C, I, K, and L: 100 Am; in D, G, H, and J: 50 Am; in E and F: 10 Am.
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mouse monoclonal anti-Ah (1:150, Dako), mouse monoclonal anti-a-synuclein (H3C, [29], J. George), rabbit polyclonal antibody which specifically recognizes tau protein with the P301L mutation (Ab P301L, [30], P. Rizzu and P. Heutink), and rabbit polyclonal antibody C-27 (recognizes the active form of calpain II, [31]). 2.4. Genetic analysis Genomic DNA was extracted either from frozen cerebellum (b3630) or from paraffin-embedded tissue (b4097). PCR amplification and sequencing was done as described in detail previously [32].
3. Results Both brains were evaluated as part of a brain donation program at the Harvard Brain Tissue Resource Center at McLean Hospital. Only a limited amount of information on family history and the course of disease was available. The first patient (b3630) was a female of North European ancestry. She presented at 53 years old with attention deficit, apathy, placidity, hyperorality, hyperphagia, and lack of insight. Initially, her memory was relatively well preserved. The disease progression was characterized by memory worsening and she died at age 65. Her mother began showing signs of dementia in her early 50s and died profoundly demented at age 72. The second patient (b4097) was a female of French Canadian ancestry who presented at age 70 with aphasia and mild dementia and died at age 75. Fresh brain weight was 925 g for b3630 and 1100 g for b4097. Both brains exhibited quite similar neuropathological findings. Macroscopically, frontal and temporal lobes were prominently atrophic, moderate atrophy was present in the remaining cerebral and brain stem structures. Substantia nigra was pale. Locus coeruleus of b3630 was well pigmented, but pale in b4097. By light microscopy, the atrophic regions showed severe neuronal loss, scattered ballooned neurons, status spongiosus (esp. upper cortical layers), and marked gliosis, prominent in superficial cortical layers and in the cortico-subcortical junction (Fig. 1A). Rare diffuse plaques and rare Pick bodies were detected in b3630. Mature neuritic plaques of Alzheimer and Lewy bodies were absent in both brains (Fig. 1B,C). A striking finding in Bielschowsky preparations were many neurons with perinuclear argyrophilic rings, in many instances with an attached small round body (Fig. 1D – F),
especially frequent in temporal and entorhinal cortices, dentate granule cells, and to a lesser extent, in the CA1 region of the hippocampus. These structures were immunopositive with anti-tau antibodies, including those that were phosphorylation-independent (tau-2), phosphorylationdependent (AT-8 and PHF-1), or conformation-dependent (MC-1) (Fig. 1G – L). Additionally, anti-tau antibodies labeled numerous astrocytic plaques, tuft-shaped astrocytes (Fig. 1L), oligodendroglial coiled bodies, and neuronal processes. Tau-immunopositivity was strong in the neocortex, amygdala, dentate gyrus, substantia innominata, substantia nigra, oculomotor nucleus, periaqueductal gray, raphe nuclei, and locus coeruleus. Direct sequencing of PCR amplified exons of the tau protein gene identified in both patients a heterozygous P301L mutation (CCG to CTG in exon 10 of tau gene, Fig. 2). A novel antibody specifically recognizing tau protein with the P301L amino acid substitution (Ab P301L) was used to determine if the aggregates of phosphorylated tau protein contain also the mutated protein [30]. On brightfield preparations (Fig. 3A – C), the staining pattern with the Ab P301L was quite similar to that observed with standard antitau antibodies (Fig. 1G – L). Multiparametric confocal microscopy utilizing double labeling with Ab P301L and PHF-1 revealed that essentially all PHF-1 positive perinuclear rings, small attached inclusions, and larger neuritic processes do indeed contain the mutated tau protein (Fig. 3D –L). The mutated protein was also present in the short thread-like structures in the neuropil, even though the PHF1 labeled threads were slightly more numerous. In both patients, immunohistochemistry for ubiquitin (Fig. 4A – C) showed a complete absence of labeling of the tau-positive rings, small inclusions, and neuritic processes, even though a rare tangle of Alzheimer in the entorhinal cortex was clearly labeled (Fig. 4A). This finding points to a possible role of abnormalities in proteolytic mechanisms as a putative mechanism of neurodegeneration in this tauopathy. Previous studies have established that neurofibrillary tangles and neuropil threads in Alzheimer’s disease label with active site-directed antibodies to calpain II (C-18 and C-27, [31]), one of the isoforms of a family of calcium-dependent cysteine proteases, an important proteolytic system involved in a variety of cellular functions. With light microscopy, the antibody C-27 labeled neurofibrillary tangles and dystrophic neurites in preparations from Down’s syndrome brain (Fig. 4D, male 42 years). In P301L tauopathy, the perinuclear rings, dystrophic processes, and rare Pick bodies were also labeled (Fig. 4E,F). In both P301L
Fig. 3. Immunohistochemistry with an antibody specifically recognizing tau protein with the P301L mutation (Ab P301L) evaluated in light microscopy demonstrates the presence of the mutated protein in many perinuclear rings in dentate granule cells (A), pyramidal neurons in parahippocampal gyrus (B), and Alzheimer-type tangles in CA2 (C). The colocalization (appears yellow in overlay images) of the mutated (Ab P301L, red) and hyperphosphorylated (PHF-1, green) tau protein in larger aggregates and dystrophic neurites was directly demonstrated by triple-label confocal immunofluorescence (D – L). Smaller neuropil threads were positive mostly for PHF-1. The images were acquired with a Leica TCS NT confocal system and represent single sections through the middle of the preparation. D – F are from dentate granule cells, G – I are from pyramidal neurons in the parahippocampal gyrus, and J – L are from CA1. In F, I, and L, the merged green and red images were overlaid with blue representing nuclear staining. Bar in A – C: 50 Am; in D – L: 25 Am.
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Fig. 4. Immunohistochemistry for ubiquitin (A – C) demonstrates absence of labeling of the tau protein aggregates in the entorhinal cortex (A) and dentate granule cells (B – C) even though a rare tangle of Alzheimer is clearly labeled (A). Antibody to the active form of calpain II (C-27) labels tangles and dystrophic neurites in Down’s syndrome (D, positive control for C-27 immunohistochemistry). In P301L tauopathy, the active form of calpain II colocalizes with PHF-1 positive perinuclear aggregates and some dystrophic neurites, both in light (E – F) and confocal (G – I) microscopy.Bar in A – F: 50 Am; in G – I: 25 Am.
brains, the evaluation by double-label confocal immunofluorescence with PHF-1 and C-27 (Fig. 4G – I) directly demonstrated colocalization of the active form of calpain II in the PHF-1 positive aggregates.
4. Discussion Extensive literature exists on clinical, pathological, and biochemical findings in the P301L tauopathy [17,18,30,32–
37]. The overall pathological features of the two brains studied in this report were consistent with previous descriptions. There were, however, several features of considerable interest, relating to both the differential diagnosis and the pathogenesis of this disorder. To further extend our knowledge about the pathophysiology involved, in addition to standard neuropathological evaluation of tauopathies, we have focused on two important issues. First, we have addressed the question of whether the aggregates of hyperphoshorylated tau do indeed contain
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the mutated protein. Both brains exhibited a striking perinuclear aggregation of argyrophilic, hyperphosphorylated tau proteins in the form of a ring, frequently with an attached small inclusion. These were especially prominent in dentate granule cells, and to a lesser extent in CA1, entorhinal, and temporal cortices. Utilizing a novel antibody specifically recognizing tau protein with the P301L mutation, we have directly demonstrated that both the rings and the attached inclusions contain the mutated protein. The finding of the mutated protein in dentate granule cells is of special significance. First, in FTD cases with prominent frontotemporal atrophy, the evaluation of dentate granule cells focuses on establishing the presence or absence of inclusions, Pick bodies being the most common. The small inclusion described here could be called a mini Pick-like body and is possibly considered to represent an early stage of the formation of a classical Pick body. However, since Pick bodies contain only 3-repeat tau isoforms [38,39], the presence of tau protein with the P301L mutation (4-repeat tau) clearly points to important biochemical differences between these types of inclusion bodies, a finding of direct relevance in the differential diagnosis of Pick’s disease. Second, since dentate granule cells normally express only 3-repeat tau isoforms [40], the presence of the mutated protein indicates an abnormality in mRNA splicing. This finding is of potentially high importance in the understanding of the pathophysiology of tauopathies in general, since it has been recently described also in the dentate fascia in Alzheimer’s disease and progressive supranuclear palsy [41]. The second important issue addressed in this report was the morphological assessment of proteolytic systems in P301L tauopathy. Abnormalities in proteolysis are being considered as one of the putative mechanisms of neurodegeneration in tauopathies [16]. Recent studies indicate that the calcium-dependent cysteine protease calpain [42] is involved in several important ways in tau protein hyperphosphorylation and neurodegeneration in general [43 – 45]. The regulatory subunit p35 of cyclin-dependent kinase 5 (Cdk5), one of the kinases involved in tau phosphorylation, is cleaved by calpain to p25. Binding of p25 to Cdk5 constitutively activates Cdk5, changes its cellular location, and alters its substrate specificity. Based on these studies, it is quite possible that the association of the active form of calpain II (C-27) with PHF-1 labeling reflects Cdk5 activation, one of the protein kinases implicated in tau hyperphosphorylation. An alternative explanation for the C-27/ PHF-1 colocalization would be the involvement of calpain in the proteolysis of tau protein. Tau incorporated into paired helical filaments (PHFs) is considerably more resistant to proteolysis by calpain [46], which could explain both the persistence of these structures and also the prominent calpain activation as a persistent attempt to degrade the abnormal protein. A recent report [47] also indicates that, at least in biochemical studies, tau proteins with the mutations V337M or R406W are less susceptible than either tau with the P301L mutation or corresponding wild-type tau to
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degradation by calpain I, the other main isoform of the protease. The differences were, at least in part, due to changes in accessibility of a cleavage site located about 100 amino acids from the C-terminus. Even though the exact interpretations of these findings will depend on further biochemical studies and the availability of antibodies, these findings nevertheless point to the important role of the calpain system in the pathophysiology of this tauopathy. The ubiquitin– proteasome complex represents another proteolytic system involved in the degradation of a variety of cellular proteins [48 – 50]. The complete lack of immunostaining of the tau aggregates for ubiquitin points to a possible abnormality in this system in the P301L tauopathy. One mechanism might involve inefficient attachment of ubiquitin by conjugating enzymes. Alternatively, tau protein might be properly conjugated, targeted to proteasome, deubiquitinated, but inefficiently degraded with subsequent aggregation in the proteasome complex. Finally, incomplete penetration of the anti-ubiquitin antibody needs to be considered. Whether the tau-positive perinuclear rings do indeed represent accumulation of undegraded tau in the proteasome awaits further ultrastructural studies. The findings nevertheless indicate that further studies need to elucidate the molecular mechanisms of proteolysis in tauopathies since they might represent important pathophysiological mechanisms of neurodegeneration in this disorder. Acknowledgements We would like to thank Dr. A. Andreadis (Eunice Kennedy Shriver Center, Waltham, MA) for helpful advice. We would also like to thank Ms. Karlotta Fitch and Mr. Timothy Wheelock for help with the preparation of microscopic slides. The antibodies PHF-1 and MC-1 were kindly provided by Dr. P. Davies (Albert Einstein College of Medicine, Bronx, NY), the antibody H3C by Dr. J.M. George (University of Illinois, Urbana-Champain, IL). Drs. P. Rizzu and P. Heutink (Erasmus University, Rotterdam, The Netherlands) kindly provided the antibody P301L. This work was supported by grants from the National Institute on Aging, AG00764, AG05134, AG17617, and AG10133. The Harvard Brain Tissue Resource Center is supported in part by PHS grant MH/NS 31862. References [1] Gustafson L. Frontal lobe degeneration of non-Alzheimer type: II. Clinical picture and differential diagnosis. Arch Gerontol Geriatr 1987;6:209 – 23. [2] Neary D, Snowden JS, Northen B, Goulding P. Dementia of frontal lobe type. J Neurol Neurosurg Psychiatry 1988;51:353 – 61. [3] Spillantini MG, Bird TD, Ghetti B. Frontotemporal dementia and Parkinsonism linked to chromosome 17: a new group of tauopathies. Brain Pathol 1998;8:387 – 402. [4] Wilhelmsen KC, Lynch T, Pavlou E, Higgins M, Nygaard TG. Localization of disinhibition – dementia – parkinsonism – amyotrophy complex to 17q21 – 22. Am J Hum Genet 1994;55:1159 – 65.
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