FTDP-17 Mutations in tau Transgenic Mice Provoke Lysosomal Abnormalities and Tau Filaments in Forebrain

MCN

Molecular and Cellular Neuroscience 18, 702–714 (2001) doi:10.1006/mcne.2001.1051, available online at http://www.idealibrary.com on

FTDP-17 Mutations in tau Transgenic Mice Provoke Lysosomal Abnormalities and Tau Filaments in Forebrain F. Lim,* F. Herna´ndez,* J. J. Lucas,* P. Go´mez-Ramos, † M. A. Mora´n, † and J. A´vila* ,1 *Centro de Biologı´a Molecular “Severo Ochoa,” Universidad Auto´noma de Madrid, 28049 Madrid, Spain; and †Departamento de Morfologı´a, Facultad de Medicina, Universidad Auto´noma de Madrid, 28029 Madrid, Spain

The tauopathies, which include Alzheimer‘s disease (AD) and frontotemporal dementias, are a group of neurodegenerative disorders characterized by filamentous Tau aggregates. That Tau dysfunction can cause neurodegeneration is indicated by pathogenic tau mutations in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). To investigate how Tau alterations provoke neurodegeneration we generated transgenic mice expressing human Tau with four tubulin-binding repeats (increased by FTDP-17 splice donor mutations) and three FTDP-17 missense mutations: G272V, P301L, and R406W. Ultrastructural analysis of mutant Tau-positive neurons revealed a pretangle appearance, with filaments of Tau and increased numbers of lysosomes displaying aberrant morphology similar to those found in AD. Lysosomal alterations were confirmed by activity analysis of the marker acid phosphatase, which was increased in both transgenic mice and transfected neuroblastoma cells. Our results show that Tau modifications can provoke lysosomal aberrations and suggest that this may be a cause of neurodegeneration in tauopathies.

INTRODUCTION The tauopathies are a family of neurodegenerative diseases (reviewed in (Heutink, 2000; Lee and Trojanowski, 1999; Spillantini and Goedert, 1998; van Swieten et al., 1999) which include AD, Down’s syndrome, progressive supranuclear palsy, amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, Pick’s disease, corticobasal degeneration, and fronto-

1 To whom correspondence and reprint requests should be addressed. Fax: 34-91-397 4799. E-mail: [email protected].

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temporal dementias. In all of these disorders a prominent neuropathological characteristic is the presence of filamentous Tau inclusions in affected brain regions, raising the hypothesis that aberrant levels or forms of Tau play a causative role in neurodegeneration. In an attempt to model tauopathies, several transgenic mice carrying cDNAs encoding either the largest or the smallest central nervous system isoform of human tau have been generated (Brion et al., 1999; Gotz et al., 1995; Ishihara et al., 1999; Probst et al., 2000; Spittaels et al., 1999). Many of these lines demonstrate that a large excess of normal human Tau can provoke some of the cellular changes observed in tauopathies but is insufficient for the formation of the mature neurofibrillary aggregates observed in the human diseases. The recent finding of tau gene mutations in FTDP-17 (Goedert et al., 1998; Hutton et al., 1998; Poorkaj et al., 1998; Spillantini et al., 1998) provided evidence that defects in this gene are sufficient to provoke neurodegeneration as well as an additional strategy for generating animal models of tauopathies. Recently two groups have reported the generation of transgenic mice expressing mutant human Tau containing the P301L mutation (Gotz et al., 2001; Lewis et al., 2000). Like the previous transgenic models these mice developed some neuropathological symptoms encouragingly reminiscent of the human diseases and in one case short filaments could be observed from brains of the transgenic mice (Gotz et al., 2001). The effect of some of the individual FTDP-17 tau mutations has been analyzed by in vitro biochemical experiments using transient transfection of mutated cDNAs in cell lines (Dayanandan et al., 1999; Hasegawa 1044-7431/01 $35.00 © 2001 Elsevier Science All rights reserved.

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et al., 1998; Hong et al., 1998; Matsumura et al., 1999; Pe´rez et al., 2000). Based on their location within the microtubule-binding repeats, it can be predicted that the G272V and P301L mutations in tau both might have an effect on microtubule binding. Several studies (Hasegawa et al., 1998; Hong et al., 1998) confirm a partial loss of microtubule-binding function, providing a mechanism by which unbound Tau might be more prone to accumulate and thus facilitate aggregation. Indeed, we have shown that peptides containing the P301L mutation have a higher propensity to aggregate than those containing the wild-type Tau sequence (Arrasate et al., 1999). Another of the first described mutations in Tau (R406W) is located outside of the microtubule-binding repeats at the C-terminal end of the molecule. Recently we (Pe´rez et al., 2000) and others (Dayanandan et al., 1999; Matsumura et al., 1999) have found that in transfected cells the R406W mutation reduces phosphorylation of Tau at a site close to the C-terminus of the molecule recognized by PHF-1, an antibody traditionally considered a marker of Tau predisposed to aggregation. However, ultrastructural analysis of Tau inclusions from FTDP-17 reveals the presence of both straight filaments and paired helical filaments (PHFs) similar to those found in AD patients (Heutink, 2000). Thus it appears that reduced phosphorylation of the PHF-1 epitope near the Tau C-terminus does not necessarily correlate with reduced aggregation of Tau into filaments typical of tauopathies. Indeed we have further demonstrated that the phosphorylated mutant protein is unable to bind to microtubules, affecting microtubule dynamics (Pe´rez et al., 2000) and possibly favoring aggregation because of an accumulation of phosphorylated Tau in the cytoplasm. Finally, in several FTDP-17 families, the only tau mutations found have been those which affect the splicing of exon 10 to increase the ratio of 4-repeat to 3-repeat isoforms (Heutink, 2000). Furthermore, in the majority of FTDP-17 victims, the polymers assembled from Tau only contain the 4-repeat isoforms (Heutink, 2000). Taken together, these observations indicate that the 4-repeat forms of Tau may favor fibril formation compared to the 3-repeat forms. To test whether FTDP-17 mutations can be used to create an animal model of tauopathies, we generated transgenic mice expressing a 4-repeat isoform of Tau containing the three FTDP-17 mutations G272V, P301L, and R406W simultaneously, aiming for a cummulative effect stronger than that using any single mutation alone, similar to the strategy previously used to generate transgenic mice expressing mutant APP (SturchlerPierrat et al., 1997).

FIG. 1. VLW transgenic mice express mutated human Tau in cortex and hippocampus. (A) Diagram showing the structure of the transgene used to express the largest human CNS Tau isoform (open box) containing the two N-terminal inserts (striped boxes) and 4-microtubule-binding-repeat elements (gray boxes). The three FTDP-17-linked mutations G272V (V), P301L (L), and R406W (W) were incorporated by site-directed mutagenesis of the human tau cDNA. Neuron-specific expression was directed by insertion of the cDNA into a murine thy1 gene expression cassette (black) between exons (black boxes) 2 and 4. (B) Western blot analysis of recombinant Tau expression in VLW mice. Protein extracts from wild-type (wt) mice (1.5 months) and transgenic VLW mice at ages 1.5 and 5.5 months were probed with anti-Tau antibodies T14 and 7.51. The human-specific T14 antibody reacted with only a single band corresponding to the transgene product, while 7.51, which recognizes both human and mouse Tau, detected two major bands in the transgenic extracts, the upper corresponding to the transgene product. Extracts were prepared from cortex (Cx), hippocampus (Hp), cerebellum (Cb), striatum (St), and spinal cord (SC).

RESULTS Generation of (VLW) Mice Exhibiting High Levels of Mutant Human Tau in Hippocampus and Cortex We generated a transgenic mouse line, VLW, which expresses the human 4-microtubule-binding-repeat Tau isoform with the two N-terminal inserts derived from exons 2 and 3, together with three mutations, G272V, P301L, and R406W, which have been identified in human victims of FTDP-17. Neuron-specific expression was directed by cloning the mutated tau cDNA into a mouse thy1 promoter gene (Fig. 1A) cassette (Luthi et al., 1997) which has previously been used to express mutant human APP isoforms preferentially in hippocampus and neocortex of transgenic mice (SturchlerPierrat et al., 1997). To determine the distribution and levels of transgene expression we analyzed extracts from microdissected cortex, hippocampus, cerebellum, striatum, and spinal cord by Western blotting (Fig. 1B) with the humanspecific anti-Tau antibody T14. These data show that

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FIG. 2. Tau immunohistochemistry in VLW mice. (A–G) Immunohistochemistry with human-specific anti-Tau antibody T14 of sagittal sections of the hippocampus (A, B, C, and F) and cortex (D, E, and G). (A–C) Hippocampi from 1.5-month-old wild-type (A) and transgenic (B) and 10-month-old transgenic (C) mice showing high transgene expression in the CA1, CA3, and hilus regions. Bar, 0.5 mm. (D and E) Cerebral cortex from 1-month-old wild-type (D) and transgenic (E) mice. Numerous cortical neurons show intense immunoreactivity for human Tau in transgenic mice. Bar, 0.5 mm. (F) CA1 region from a 10-month-old transgenic mouse showing accumulation of recombinant Tau in the cell bodies as well as the processes of hippocampal neurons. Bar, 0.5 mm. (G) Detailed view of intensely stained cortical neurons from a 10-month-old transgenic mouse showing dystrophic neurites (arrows). Bar, 0.1 mm. (H and I) Immunohistochemistry with phosphorylation-sensitive antibody AT180 of the CA1 hippocampal region of 10-month-old wild-type (H) and transgenic (I) mice. Bar, 0.1 mm. (J–L) Phospho-Tau immunohistochemistry of cortical sections. Bar, 0.1 mm. Neurons in 10-month-old wild-type mice (J) exhibited little staining with antibody AT180, while strong somatodendritic staining was observed in 10-month-old VLW mice (K). AT8 staining revealed a similar pattern in cortical neurons from 5-month-old mice (L).

VLW mice but not wild-type controls express recombinant mutant Tau at high levels in cortex and hippocampus, moderately in striatum, weakly in spinal cord, and

not at all in cerebellum (Fig. 1B, top panel). Using the antibody 7.51 (Fig. 1B, bottom panel), which recognizes both human (top band) and murine (bottom band) Tau

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FIG. 3. Tau filaments in forebrain of VLW mice. (A) Light micrograph (40⫻) from the CA1 hippocampal sector. Some of the pyramidal neurons are heavily labeled by the antibody T14 (black arrows), whereas others (empty arrows) are not immunostained. (B) Electron micrograph of one of the immunopositive neurons contained in the section shown in A. A diffuse mass of reaction product filled most of the perikaryon and the apical dendrite. N, nucleus; bar, 1 ␮m. (C) High magnification of the portion of the apical dendrite framed in B where immunopositive

706 it was apparent that recombinant Tau levels in all tissues examined except cerebellum were similar to or higher than endogenous Tau levels. The ratio of transgenic to endogenous Tau appeared to increase with the age of the transgenic mice: in cortex this ratio increased from 1.1 at 1.5 months to 2.5 at 5.5 months, while in hippocampus this increase was from 0.8 to 1.1. Immunohistochemical analysis of sagittal brain sections from VLW mice (Fig. 2) revealed intense staining of the transgene in neuronal cell bodies and neurites of the cortex and hippocampus. The spatial pattern of transgene expression observed was similar in mice of all ages examined (1–10 months). Pyramidal neurons in CA1 and CA3, but not CA2, contained high amounts of recombinant Tau (Figs. 2B and 2C), while granule cells of the dentate gyrus (Figs. 2B and 2C) and processes from CA1 (Fig. 2F) showed increased staining with the age of the mice. In cerebral cortex numerous pyramidal cells were strongly positive for the transgene (Fig. 2E) and dystrophic neurites were frequently observed (Fig. 2G). When we investigated the distribution of total Tau (i.e., both human and murine) the immunostaining pattern revealed using antibody 7.51 (not shown) was virtually identical to that of T14, with the difference that a higher basal level of staining due to endogenous murine Tau was observed in neurons. Hyperphosphorylation of Tau in VLW Mice A characteristic of all tauopathies is the strong immunoreactivity of Tau hyperphosphorylated at certain epitopes which were originally characterized in AD paired helical filaments. To determine whether Tau in our VLW mice possessed such hyperphosphorylated epitopes we next used phosphorylation-sensitive antibodies to perform immunohistochemistry (Figs. 2H– 2L). Probing of hippocampal and cortical sections using the antibody AT180 directed against phosphothreonine 231 in Tau (Goedert et al., 1994) revealed that levels of this phosphoepitope were elevated (compare Figs. 2I with 2H and 2K with 2J) in the same regions where transgene expression was detected. Immunohistochemistry using antibody AT8, directed against phosphoserines 199/202 (Biernat et al., 1992; Mercken et al., 1992)

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also yielded a similar staining pattern in cortex (Fig. 2L) but not in hippocampus. Western blotting with AT8 and AT180 (not shown) confirmed results obtained in other tau transgenic mice (Brion et al., 1999; Ishihara et al., 1999; Spittaels et al., 1999) that both the transgenic and endogenous Tau proteins were phosphorylated at these epitopes. Densitometric analysis of the endogenous mouse Tau band in Western blots revealed a relative increase in the AT8/7.51 ratio of 10-month-old VLW mice compared to age-matched wild-type controls in cortex (0.48 ⫾ 0.10) and hippocampus (1.85 ⫾ 0.53). Using antibody PHF1, which recognizes phosphoserines 396/404 (Otvos et al., 1994) we did not detect any staining differences between wild-type and transgenic tissues (not shown) but this was not surprising in view of our previous results showing that the R406W mutation decreases phosphorylation of this epitope (Pe´rez et al., 2000). Tau Filaments in Cortex and Hippocampus of VLW Mice The high concentration of hyperphosphorylated somatodendendritic Tau in specific populations of neurons of VLW mice resembles the accumulation of Tau in tauopathies such as frontotemporal dementias and AD, and thus it would be expected to favor the formation of fibrillary deposits. Indeed, ultrastructural analysis of highly T14-positive neurons (Fig. 3A) revealed dense Tau immunoreactivity in apical dendrites (Figs. 3B–3D), with isolated filaments also present in the perikaryon of these immunostained neurons (Fig. 3E). Immunostaining for neurofilaments with antibody SMI-31 was negative in these Tau-reactive areas. We next prepared Sarkosyl-insoluble protein from microdissected forebrains of VLW mice and agematched controls and performed electron microscopy of negatively stained samples. In samples from transgenic mice (Fig. 3F) but not from their wild-type counterparts, long filaments were observed, confirming the above observation of Tau filaments in situ by ultrastructural analysis. Western blot analysis of Sarkosyl-insoluble transgenic extracts confirmed Tau immunoreactivity with antibody 7.51 and the filaments could also be

filaments (arrows) are observed. Bar, 0.5 ␮m. (D) Apical dendrite of a pyramidal neuron from the cerebral cortex showing T14-immunopositive filaments (arrows). Bar, 1 ␮m. (E) Perikaryal portion of an immunopositive neuron from the CAl hippocampal sector showing several T14-immunopositive filaments (arrows) and two complex lysosomal bodies (stars) with electron-dense and electron-lucent portions. Bar, 0.5 ␮m. (F) Protein filaments found in Sarkosyl-insoluble pellets extracted from forebrain of a 3-month-old VLW mouse. Various filament types can be observed (arrows): (top) thin single filaments of widths varying from 2 to 5 nm; (middle) paired entwined filaments; (bottom) thick filaments of approximately 8 nm. Scale bar represents 100 nm.

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FIG. 4. Lysosomal abnormalities in VLW mice. (A–D) Enzymatic histochemical staining for acid phosphatase in 1.5-month-old wild-type (A and C) and VLW (B and D) mice. Increased staining in transgenic samples was especially obvious in the CA1 hippocampal sector (arrows in A and B) and pyramidal neurons of the cortex (C and D). Bars, 0.5 mm. (E) Assay of soluble acid phosphatase activity in cortical, hippocampal, and cerebellar extracts derived from 10-month-old wild-type (unshaded bars) and transgenic (shaded bars) mice. Enzyme activity is expressed relative to total protein concentration. Error bars represent the standard error of the mean of three measurements.

decorated by immunogold staining with the anti-Tau antibody BR134, which also decorates paired helical filaments prepared from human AD brains (not shown). Three types of filaments could be discerned: thin filaments of diameters ranging from 2 to 5 nm (Fig. 3F, top), twisted filaments apparently composed of two intertwined thin filaments (Fig. 3F, middle), and thick straight filaments approximately 8 nm in diameter (Fig. 3F, bottom). Forebrain Lysosomal Abnormalities in VLW Mice At the ultrastructural level we also noted an increase in the number of lysosomal complexes in neurons of VLW mouse brains compared to wild-type animals of the same age. In Tau-positive neurons many single and complex lysosomal bodies with electron-dense and electron-lucent elements could be observed (Fig. 3E). No surrounding membrane was seen in many of these

complex bodies (Fig. 3E), indicating that they could be residual bodies derived from lysosomes. These complex lysosomal bodies were also found, but less often in some Tau-immunonegative neurons. Interestingly, ultrastructural analysis revealed these lysosomal differences between 1- and 1.5-month-old wild-type and transgenic mice in the absence of Tau filaments. To confirm our observation by electron microscopy that neurons in VLW mice show lysosomal abnormalities, we carried out enzymatic activity staining for acid phosphatase, a classical marker of lysosomes (e.g., see de Duve et al., 1962). A comparion of transgenic VLW mice with age-matched wild-type controls showed an increase in staining intensity in the cortex and hippocampus (Fig. 4). The most evident increases were observed in pyramidal neurons of the cortex (Figs. 4A and 4B) and the CA1 hippocampal sector (Figs. 4A and 4B) in a staining pattern very similar to that produced by the anti-Tau antibody T14 (see Fig. 2). In the cere-

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FIG. 5. SHSY5Y neuroblastoma cells stably transfected with VLW mutant tau have increased acid phosphatase activity. (A) Enzymatic histochemical staining for acid phosphatase in SHSY5Y cells: control untransfected (con) or transfected with wild-type tau cDNA (wt) or VLW mutant tau cDNA (VLW). Left panels show low-magnification views of the total pool of stably transfected cells. Bar, 0.2 mm. Right panels show high-magnification views of individual representative cells from each pool. Bar, 0.1 mm. (B) Assay of soluble acid phosphatase activity in extracts derived from the SHSY5Y pools shown in A. The mean enzyme activity normalized with respect to cell number is expressed as a ratio with respect to activity measured in untransfected SHSY5Y cells (set at 1). Error bars indicate the standard error of the mean of three measurements. (C) Western blots showing Tau protein as detected by the antibody T14 (top panel, Tau) and ␤-tubulin (bottom panel, tub) in each of the SHSY5Y pools shown in A. In untransfected SHSY5Y cells the smallest Tau isoform (bottom band) predominates, while in the stable transfectants the major proportion of Tau expressed is the largest central nervous system isoform (top band) encoded by the transgene. The histogram below compares Tau levels normalized with respect to tubulin levels (Tau/tubulin). Error bars represent standard error of mean values, which are expressed with respect to the normalized Tau level in untransfected SHSY5Y cells set at 1.

bellum (not shown) acid phosphatase staining differences between transgenic and wild-type animals were not observed. These results were confirmed quantitatively by assaying acid phosphatase activity in extracts made from cortex, hippocampus, and cerebellum from wild-type and transgenic VLW mice. As shown in Fig. 4E the specific activity of acid phosphatase (per milligram of total protein) was increased in transgenic cortical and hippocampal extracts but not in cerebellar extracts compared to wild-type controls. The VLW Mutations in Tau Increase Acid Phosphatase Activity in Stably Transfected SHSY5Y Neuroblastoma Cells Since we had observed lysosomal disturbances in the brains of VLW mice, we next investigated whether this is provoked by increased neuronal Tau levels or by the presence of the mutations. That the latter is the major contributing factor is suggested by the fact that abnormal lysosomes have not been reported in any of the

mice transgenic for wild-type human tau characterized so far (Brion et al., 1999; Duff et al., 2000; Gotz et al., 1995; Ishihara et al., 1999; Probst et al., 2000; Spittaels et al., 1999). We directly compared wild-type and mutant VLW tau by selecting stable SHSY5Y neuroblastoma transfectants of the two cDNAs cloned into plasmids conferring neomycin resistance. By analyzing the total pool of transfectant clones resistant to the neomycin analogue G418, integration site-specific effects were ruled out. In Fig. 5B it can be seen that acid phosphatase activity is increased in extracts made from cells transfected with the VLW mutant compared to those transfected by wild-type tau or untransfected cells. This result was confirmed by cytochemical staining for acid phosphatase activity, which was more intense in the VLW transfectants than wild-type tau transfectants or untransfected SHSY5Y (Fig. 5A, left panels). Western blots of the cell extracts (Fig. 5C) indicated that levels of total Tau were increased in the wild-type tau and VLW transfectants since a higher Tau/tubulin ratio was detected in these cells than in untransfected SHSY5Y cells.

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Curiously, while untransfected SHSY5Y cells express mainly the smaller isoforms of tau, the selection of the wild-type tau and VLW transfectants, which contain cDNAs encoding the largest central nervous system tau isoform, resulted in downregulation of the lower molecular weight isoforms. A likely explanation for this observation is that total Tau levels probably cannot exceed a certain limit without interfering with cell division and growth, and thus transgene expression is possible only in cells with lower endogenous levels of Tau. Importantly, since the total Tau level in the VLW transfectants was similar to (and even slightly less than) that of the wild-type tau transfectants (Fig. 5C, histogram), the increase in acid phosphatase activity detected in the latter can be attributed to the VLW mutations and not merely to higher levels of Tau in these cells. Closer inspection of fixed cells stained for acid phosphatase activity also revealed differences in the distribution of lysosomes in VLW-transfected cells: staining in normal SHSY5Y cells or those transfected by wild-type tau was usually perinuclear and confined to a semilunar patch, whereas staining in the VLW transfectants was generally widespread and covered most of the cell (Fig. 5A, right panels).

DISCUSSION The pathological hallmarks of AD, the most frequent form of dementia, are cortical and hippocampal lesions displaying neurofibrillary tangles (NFTs) of Tau protein and amyloid plaques of A␤ peptides. Although the temporal and spatial distribution of NFTs best correlates with observed neurodegeneration (Arriagada et al., 1992), curiously no genetic defects in the tau gene have yet been linked with AD. Notwithstanding, other tauopathies are characterized almost exclusively by inclusions of aggregated Tau (for reviews see Heutink, 2000; Lee and Trojanowski, 1999; Spillantini and Goedert, 1998; vanSwieten et al., 1999) and do not exhibit other lesions, such as A␤ deposits. Tau found under such pathological conditions is characteristically hyperphosphorylated and aggregated into filamentous deposits. In this study we have generated mice transgenic for a mutant human tau isoform encoding the three amino acid changes G272V, P301L, and R406W which have been found in FTDP-17 kindreds (Hutton et al., 1998). These VLW mice develop Tau filaments preferentially in the forebrain, whereas all previous mice transgenic for tau cDNAs (Brion et al., 1999; Gotz et al., 2001, 1995; Ishihara et al., 1999; Lewis et al., 2000; Probst et al., 2000; Spittaels et al., 1999) show less localized pathology in

709 the brain, with large accumulations of Tau extending as far as the spinal cord. Consequently, in all of these models where behavioral studies were conducted, motor deficits were noted. Transgene expression in our VLW mice is low in spinal cord, and consistent with this observation, VLW mice up to 12 months old do not demonstrate obvious motor abnormalities. Aberrations are observed in a subset of hippocampal and cortical neurons compatible with the neuronal populations at risk to degenerate in human tauopathies. A possible reason for the more restricted distribution of Tau pathology in our VLW mouse line may be due to the lower levels of transgene expression (up to 2.5-fold compared to endogenous murine Tau) in our mice compared to the massive overexpression (up to 15-fold) observed in others (Ishihara et al., 1999). Lower overall expression probably renders the “leakage” from the thy1 promoter to negligible levels in regions where the promoter is normally not active. On the other hand, it is likely that with the incorporation of pathogenic mutations in our VLW mice, neurotoxic effects can be observed at lower Tau protein levels. This is supported by the fact that in transgenic mice expressing lower levels of wild-type human Tau either from a cDNA minigene (Gotz et al., 1995) or from human genomic DNA containing coding sequences, introns, and regulatory regions (Duff et al., 2000), fibrillary aggregates are not observed. In mice containing genomic human tau, the transgene product was absent from neuronal cell bodies and restricted to neurites and synapses, indicating that mislocalization of Tau expressed from cDNA minigenes due to the lack of appropriate genomic targetting elements (Behar et al., 1995; Litman et al., 1993) may be yet another pathological factor in transgenic animal models. Similar to observations in other tau transgenic mice, the presence of the recombinant mutant Tau in our VLW mice also appears to alter either the intracellular kinase/phosphatase balance, and/or the interaction of these enzymes with their target motifs, since neurons in regions containing high levels of transgene are also strongly stained by the phosphorylation-dependent anti-Tau antibodies AT180 (specific for phosphothreonine 231) and AT8 (specific for phosphoserines 199/ 202). Thus it appears that this aspect of the human diseases is recapitulated in VLW mice since these epitopes are known to be hyperphosphorylated in tauopathies (reviewed in Spillantini and Goedert, 1998). At the ultrastructural level, we observed a progression of intraneuronal accumulation of Tau in neurons of the cortex and hippocampus of our VLW mice from diffuse and patchy Tau staining in young mice to long filaments in older mice. Isolation of Tau filaments in

710 vitro revealed fine single filaments, paired entwined fine filaments, and thick filaments, suggesting that these may also represent progressive stages in the neurofibrillary aggregation of Tau. Once formed, these polymers may act as seeds for the further recruitment of cytosolic Tau into the insoluble pool. Additionally, the aggregation of insoluble Tau has also been proposed to contribute to hyperphosphorylation by the trapping of phosphate groups in a form inaccessible to protein phosphatases (Billingsley and Kincaid, 1997). In this way, mutant Tau may be seen to act in a prion-like fashion, inducing biochemical changes in endogenous Tau molecules which, once altered, can also serve to provoke further changes. This suggestion agrees well with previous in vitro experiments demonstrating the sequestering of normal soluble Tau by abnormal Tau extracted from Alzheimer’s disease tissue (Alonso et al., 1996, 1994). The presence of large amounts of aggregated protein is likely to pose a problem for the cellular waste disposal system. As a result of aging or metabolic and oxidative stress such indigestible material accumulates within lysosomal compartments, giving rise to a heterogenous group of vesicular bodies with electron-dense contents (Brizzee et al., 1975). This process has been shown to be accelerated in neurodegenerative conditions such as Alzheimer’s disease together with the proliferation of lysosomes and activation of lysosomal enzymes in neuronal regions where cell loss occurs (Cataldo et al., 1996, 1994; Hof, 1997). This situation appears to be mimicked in our VLW mice, where the lysosomal marker acid phosphatase is upregulated in neuronal populations located in regions which accumulate filamentous Tau aggregates. Our results with transfected neuroblastoma cells indicate that the VLW mutations in tau provoke the activation of lysosomal enzyme activity, providing evidence that the mutant gene product is more pathogenic than the normal protein. This change was observed in our mice at an early age, when Tau filaments were still not apparent, indicating that acid phosphatase may be a useful marker for early stages of human tauopathies such as FTDP-17. Further study will be necessary to determine if the abnormalities are due to activation of the endosome–lysosome pathway or direct perturbation of lysosomes. Our finding that tau mutations can disrupt lysosomal function in transgenic mice highlights the nature of the link between this microtubule-binding protein and organelle regulation. Overexpression of tau in cultured cells has been shown to affect the microtubule-dependent transport of organelles (Ebneth et al., 1998; Trinczek et al., 1999), while the lysosomal pro-

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tease cathepsin D could be involved in the processing of Tau into precursors of neurofibrillary tangles (Bednarski and Lynch, 1996; Bi et al., 2000). The connection between tauopathies such as FTDP-17 and lysosomal aberrations has not yet been investigated, but recent studies indicate that the vulnerable neuronal populations in Alzheimer’s disease are highly prone to lysosomal disturbances (Cataldo et al., 1996). It appears that these can be triggered in specific neuronal regions with strikingly similar spatial distributions by various means, e.g., during normal aging, metabolic, and oxidative stress and experimentally by lysosomal protease inhibitors (Bi et al., 1999), overexpression of neurotoxic APP fragments (Oster-Granite et al., 1996), and, as shown in the present study, by expression of a mutant Tau protein. Interestingly, in our stably transfected SHSY5Y cells, the intracellular distribution of lysosomes also appears to be altered. Previous results demonstrating the influence of Tau on microtubule-mediated transport (Ebneth et al., 1998; Trinczek et al., 1999) offer an explanation for this observation. In wild-type neurons Tau competes with kinesin for binding sites on microtubules such that regulation of Tau levels within the cell limits the extent of plus-end-directed (outbound) transport along microtubules (Trinczek et al., 1999). Organelles such as lysosomes thus cluster toward the cell center. In neurons expressing Tau with pathological mutations, the cytosolic pool of Tau is increased by the added presence of mutant Tau which binds poorly to microtubules. The increased concentration of free Tau favors aggregation into filaments, which in turn depletes the soluble pool, thus shifting the bound/ unbound equilibrium to release more Tau from microtubules. In addition, a fraction of the microtubulebound Tau is recruited into the cytosolic pool due to increased phosphorylation provoked by the presence of the mutant Tau. The sum result of these changes is less Tau bound to microtubules, diminishing the inhibition of kinesin-dependent transport and resulting in dispersion of lysosomes throughout the cell. Additionally, we have observed that the presence of mutated Tau also provokes dysregulation of lysosomes (e.g., upregulation of lysosomal enzymes) by an unknown mechanism. Disruption of this link may be a good therapeutic target since lysosomal disturbances may result in destructive consequences, such as autolysis or axotomy in neurodegenerative conditions (Bednarski et al., 1997). Our VLW mice and stably transfected neuroblastoma cells represent good model systems in which to investigate this theme.

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Lysosomes and Fibrils in FTDP-17 tau Transgenic Mice

EXPERIMENTAL METHODS Generation of Transgenic Mice The plasmid pSGT42 (Montejo de Garcini et al., 1994) which encodes a human 4-repeat tau isoform with two N-terminal exons was used as a template to introduce the FTDP-17 mutations G272V and P301L separately with the Quikchange (Stratagene) procedure. A triple mutant tau cDNA was then assembled by ligation of the restriction fragments SacII/AseI (containing the G272V mutation) and AseI/HindIII (containing the P301L mutation) into the plasmid pSGTR406W (Pe´rez et al., 2000) previously cut with SacII/HindIII to give rise to the plasmid pSGTVLW. The mutant tau open reading frame was excised from pSGTVLW as a BamHI/BglII fragment and ligated into the BamHI site of pBKCMV (Stratagene) in the forward orientation with respect to the CMV promoter to produce pBKVLW. The SalI/XhoI fragment of pBKVLW was then ligated into the XhoI site of pTSC21k (Luthi et al., 1997) in the forward orientation with respect to the thy1 pormoter. The resulting plasmid pTTVLW was confirmed to encode the three specific amino acid changes G272V, P301L, and R406W by sequencing of the SacII–HindIII region. Vector sequences were eliminated by NotI digestion and gel purification of the large fragment, which was then introduced by pronuclear injection into single-cell CBA ⫻ C57BL/6 embryos. Founder mice were identified by PCR and crossed with wild-type C57BL/6 mice. All transgenic mice analyzed were heterozygotes. Mice were housed four per cage with food and water available ad libitum and maintained in a temperature-controlled environment on a 12/12 h light– dark cycle, with light onset at 07:00 h. PCR screening was performed on tail DNA using the oligonucleotides TT1, 5⬘-CTCTGCCCTCTGTTCTCTGG-3⬘ (in exon 2 of the murine thy1 gene); TT2, 5⬘-CCTGTCCCCCAACCCGTACG-3⬘ (at the 5⬘ end of the human tau cDNA); and THY, 5⬘CGCTGATGGCTGGGTTCATG-3⬘ (in intron 2 of the murine thy1 gene). We used TT1 and TT2 to amplify a 470-bp product specifically from the transgene and not from endogenous murine DNA, while as an internal control for DNA, TT1 and THY were used to amplify a 450-bp product specifically from murine genomic DNA but not from the transgene.

ligated into the BamHI site of pBKCMV (Stratagene) in the forward orientation with respect to the CMV promoter to produce pBKT42. This plasmid and its triple mutant counterpart pBKVLW (see above) were transfected into SHSY5Y human neuroblastoma cells (Biedler et al., 1978) with Lipofectamine (Gibco-BRL) according to the manufacturer’s instructions and stable transfectants were obtained by selection for 1 month in 0.8 mg/ml of geneticin (G418 sulfate, Gibco-BRL). Immunochemistry Antibodies used to detect Tau were T14 (Zymed Laboratories, Inc.), which recognizes amino acids 141–178 of human Tau but fails to recognize murine Tau (Merrick et al., 1996); 7.51 (Dr. C. M. Wischik), which is directed against the microtubule-binding region (Novak et al., 1991); AT8 and AT180 (Innogenetics), which respectively recognize phosphorylated Tau at phosphoserines 199/202 (Biernat et al., 1992; Mercken et al., 1992) and phosphothreonine 231 (Goedert et al., 1994); PHF-1 (Dr. P. Davies), which recognizes Tau isoforms when phosphorylated at serines 396 and 404 (Otvos et al., 1994); and BR134 (Dr. C. M. Wischik) directed against the C-terminus of Tau. A monoclonal antibody directed against ␤-tubulin (Sigma No. T4026) was used as an internal control for protein quantity. For tissue analysis mice were sacrificed with CO 2 and brains were quickly dissected out onto an ice-cold plate. One half was processed for Western blotting, while the other half was processed for immunohistochemistry. Western blotting and immunohistochemistry were performed as previously described (Lucas et al., 2001). Protein levels were quantified by densitometry of three exposures of each of two separate Western blot experiments. Primary antibody dilutions were T14 (1/2000), 7.51 (1/100), PHF-1 (1/150), AT180 (1/50), and AT8 (1/50). T14- and 7.51immunostained tissue sections were processed for electron microscopy as previously described (Lucas et al., 2001). Protein extracts prepared from SHSY5Y cells and stable transfectants thereof were processed and analyzed by Western blot using anti-Tau antibody T14 (1/2000) and anti-␤-tubulin antibody T4026 (1/1000). Tau levels were normalized with respect to tubulin and mean values were calculated from densitometry of three exposures of each of two separate Western blot experiments.

Stable Neuroblastoma Transfectants The human wild-type tau open reading frame encoding the 4-microtubule-binding-repeat isoform with two N-terminal inserts was excised from pSGT42 (Montejo de Garcini et al., 1994) as a BamHI/BglII fragment and

Analysis of Sarkosyl-Insoluble Filaments Preparation of Sarkosyl-insoluble extracts from mouse brain and electron microscopy of filaments was carried out as described previously (Perez et al., 1998). Rabbit

712 anti-Tau serum BR134 (a kind gift from Dr. C. M. Wischik, Medical Research Council, Cambridge, UK) was used at a dilution of 1/20 for immunoelectron microsopy. Detection of Acid Phosphatase Activity and Histochemical Staining Acid phosphatase activity was assayed as described previously (Moss, 1984) by using 4-nitrophenyl phosphate as substrate in 37.5 mM citrate buffer, pH 4.9. Extracts were prepared from different brain areas and cell cultures by homogenization in 20 mM Hepes, pH 7.4, 100 mM NaCl, and 1% Triton X-100. Acid phosphatase activities in SHSY5Y cells and stable transfectants were normalized with respect to cell number and values were expressed relative to the acitvity in untransfected SHSY5Y cells set at 1. Histochemical staining for acid phosphatase activity was carried out using napthol AS-BI phosphate and Fast garnet GBC (Kit 181-A, Sigma) according to the manufacturer’s instructions.

ACKNOWLEDGMENTS We thank H. van der Putten for the plasmid pTSC21k, C. M. Wischik for antibodies 7.51 and BR134, and P. Davies for antibody PHF1. We are grateful to G. Morata for the use of microscope and photography facilities, M. Rejas and M. Arrasate for help with Tau filament electron microscopy, E. Champion and E. Langa for technical assistance, and the personnel of the Transmission Electron Microscopy Service of the UAM-SIDI for their excellent work. We thank J. Diaz-Nido, F. Wandosell, and T. Schimmang for critical reading of the manuscript and helpful discussions. This work was supported by grants from Sanofi-Synthelabo, the Comunidad de Madrid, the Fundacion “La Caixa,” and the Spanish CICYT and by an institutional grant from the Fundacio´n Ramo´n Areces. J.J.L. was supported by a reincorporation contract from the Spanish Ministry of Education and Culture.

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