Human misfolded truncated tau protein promotes activation of microglia and leukocyte infiltration in the transgenic rat model of tauopathy

Journal of Neuroimmunology 209 (2009) 16–25

Contents lists available at ScienceDirect

Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m

Human misfolded truncated tau protein promotes activation of microglia and leukocyte infiltration in the transgenic rat model of tauopathy Norbert Zilka a,b, Zuzana Stozicka b, Andrej Kovac a,b, Emil Pilipcinec c, Ondrej Bugos b, Michal Novak a,b,c,⁎ a b c

Institute of Neuroimmunology, Slovak Academy of Sciences, Dubravska 9, 845 10 Bratislava, Slovak Republic Axon Neuroscience GmbH, Rennweg 95b, 1030 Vienna, Austria Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine, Komenskeho 73, 041 81 Kosice, Slovak Republic

a r t i c l e

i n f o

Article history: Received 12 November 2008 Received in revised form 12 January 2009 Accepted 12 January 2009 Keywords: Neuroinflammation Neurofibrillary degeneration Truncated tau Microglia Leukocytes influx Alzheimer's disease

a b s t r a c t It has been hypothesized that misfolded tau protein could be a mediator of the inflammatory response in human tauopathies. Here we show that neurodegenerative lesions caused by human truncated tau promote inflammatory response manifested by upregulation of immune-molecules (CD11a,b, CD18, CD4, CD45 and CD68) and morphological activation of microglial cells in a rat model of tauopathy. In parallel, the innate immune brain response promotes activation of MHC class II positive blood-borne leukocytes and their influx into the brain parenchyma. These findings have important consequences for the rationale drug development of effective inflammation-based therapeutic strategies for human tauopathies. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Neuroinflammation plays a fundamental role in the progression of Alzheimer's disease (AD) and other tauopathies (McGeer and McGeer, 1995, 2004; Lukiw and Bazan, 2000; Wyss-Coray and Mucke, 2002; Mrak and Griffin, 2005). It may be triggered by the accumulation of misfolded proteins released from injured neurons and synapses (Wyss-Coray and Mucke, 2002). In Alzheimer's disease, neuroinflammation includes a wide spectrum of inflammatory molecules such as cytokines and chemokines, the proteins of the classical and alternative complement pathways, the acute-phase reactants, peroxisomal proliferators-activated receptors, components of the coagulation pathways, proteoglycans, cathepsins and cystatins, heat shock proteins, the metallomatrix proteinases and intercellular adhesion molecules (McGeer and McGeer, 1999, 2002; Tuppo and Arias, 2005). It is well documented that extracellular aggregates of beta amyloid (Aβ), which senile plaques are comprised of, are considered to be responsible for initiating the non-immune mediated chronic inflammatory response manifested by activated microglia and astrocytes (Akiyama et al., 2000; Mrak and Griffin, 2001; Walsch and Aisen, 2004; Eikelenboom et al., 2006). Beta amyloid deposits attract microglia and activate them to produce acute-phase proteins, complement components, cytokines ⁎ Corresponding author. Institute of Neuroimmunology, Slovak Academy of Sciences, Dubravska 9, 845 10 Bratislava, Slovak Republic. Fax: +421 2 5477 4276. E-mail address: [email protected] (M. Novak). 0165-5728/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2009.01.013

and chemokines (Blasko and Grubeck-Loebenstein, 2003; Blasko et al., 2004). On the other hand, the plaque-associated microglia are considered an important element in the degradation of Aβ and transformation of early diffuse amyloid deposits into the late neuritic Aβ plaques (Mrak and Griffin, 2005; Tuppo and Arias, 2005; Rogers et al., 2002; D'Andrea et al., 2004). Activated microglia are also present on and around neurofibrillary tangles at early (Sheng et al., 1997) as well as at later stages of tangle formation (Cras et al., 1991; Perlmutter et al., 1992; Dickson et al., 1996; DiPatre and Gelman, 1997; Oka et al., 1998; Overmyer et al., 1999; Sheffield et al., 2000). Further, it has been demonstrated that microglial activation was also correlated with tau burden in other human tauopathies such as tangle-predominant dementia, Guam parkinsonism dementia, progressive supranuclear palsy and corticobasal degeneration (Schwab et al., 1996; Ishizawa and Dickson, 2001; Imamura et al., 2001). Although activation of microglia linked to tau deposition has been documented in mice transgenic for human mutant tau protein (Bellucci et al., 2004; Yoshiyama et al., 2007), little is known about the inflammatory immunomolecules involved in this response. In order to decipher the pattern of neuroinflammation promoted by tau neurodegeneration, we used rats transgenic for human truncated tau protein, exhibiting characteristic features of human tauopathies including neurofibrillary tangles and progressive axonopathy (Zilka et al., 2006). These pathological changes were associated with functional impairment characterized by a variety of neurobehavioural symptoms (Hrnkova et al., 2007). In the present

N. Zilka et al. / Journal of Neuroimmunology 209 (2009) 16–25

study we tested: a) the hypothesis that neurofibrillary lesions induced by human truncated tau protein may stimulate inflammatory response; b) what immunophenotypic profile of inflammatory cells is characteristic of this process and c) whether blood born leukocytes take part in tau induced inflammatory response. 2. Materials and methods 2.1. Transgene construct and generation of transgenic animals The transgene construct was prepared by cloning a truncated human tau cDNA encoding amino acids 151–391 into the mouse Thy-1 gene immediately downstream of the brain promoter/enhancer sequence. The ATG codon follows immediately after the Ban I site at the end of the brain enhancer. The original Thy-1 gene sequence coding for exons II to IV, together with the thymus enhancer sequence was replaced by the human cDNA sequence. The cloned construct was introduced into DH5α bacteria and amplified. Plasmid DNA was purified using EndoFree Plasmid Max Kit (Qiagen, Hilden, Germany). Transgenic DNA was linearized by EcoRI and resolved by electrophoresis on SeaPlaque GTG agarose gel (Cambrex, Rockland, ME). The fragment of plasmid DNA encoding the transgene (which lacked any prokaryotic sequences) was extracted using QIAquick Gel Extraction Kit (Qiagen) and finally dissolved in microinjection buffer (Tris–EDTA pH 7.5). Transgenic rats were generated by pronuclear injection of one-day old rat embryos (strain SHR), which were implanted into pseudopregnant females (strain Wistar). Founders were double screened by PCR using Thy1-specific and tau-specific primers that hybridized to sequences flanking the start and stop codons of the transgene (forward: 5′-GTGGAT CTCAAGCCCTCAAG-3′, reverse: 5′CCTGATTTTGGAGGT-3′, forward: 5′-GGTGA CCTCCAAGTGTGG-3′, reverse: 5′-TATGCATGGAGGGAGAAG-3′). Primers that hybridized to the rat endogenous tau gene were used as an internal amplification control. 2.2. Animals The study was performed with 6–8 month-old transgenic rats (transgene line SHR72) expressing human truncated tau (Axon Neuroscience, Vienna, Austria) and age-matched wild-type SHR rats (Aitman et al., 1999). Rats were housed in a temperature and humidity-controlled environment with food and water made available ad libitum. The experiments were performed in accordance with the Slovak and European Community Guidelines, with the approval of the Institute's Ethical Committee. All experiments were approved by the State Veterinary and Food Administration of the Slovak Republic.

17

60 min the sections were fixed with acetone/ethanol fixative solution for 10 min at 4 °C. 2.5. Frozen section method with prefixation Animals were perfused with PBS, and the brains removed. Tissue was postfixed in 4% paraformaldehyde for 4 h, followed by treatment with 25% sucrose for 48 h to provide cryoprotection. Material was then frozen in isopentane at −40 °C. Coronal sections (40 μm) were cut in a cryostat at −18 °C. Free-floating sections were used for immunofluorescence studies. 2.6. Immunohistochemistry Immunohistochemical analyses were performed on adjacent paraffin sections using a panel of antibodies as described in Table 1. For single-labeling experiments sections were incubated for 20 min at room temperature in PBS containing 0.3% Triton X-100 and 1% H2O2, followed by a 30-minute incubation in blocking solution (PBS, Table 1 A list of immunohistochemical reagents used in this study. Antibody clone

Species

Working Specificity dilution

Vendor

W3/25 (anti-CD4)

Mouse

1:300

Serotec

OX-8 (anti-CD8)

Mouse

1:300

WT1 (anti-CD11a)

Mouse

1:300

OX-42 (antiCD11b) ED8 (anti-CD11b/ CD18)

Mouse

1:300

Mouse

1:300

6G2 (anti-CD18)

Mouse

1:300

8A2 (anti-CD11c)

Mouse

1:50

OX-1 (anti-CD45)

Mouse

1:300

ED1 (anti-CD68)

Mouse

1:300

OX-6 (anti-RT1B)

Mouse

1:300

Ox-17 (anti-RT1D)

Mouse

1:300

OX-62 (anti-alpha E2) AT8 (antiphosphotau) AT180 (antiphosphotau) PolyTau (anti-tau)

Mouse

1:300

Mouse

1:1000

Mouse

1:1000

2.3. Tissue preparation Paraffin method. Rats anesthetized with xylasine and ketamine were perfused transcardially with phosphate buffered saline (PBS; 0.01 M, pH 7.4) followed by 4% paraformaldehyde. Brains were removed immediately following transcardial perfusion and postfixed in 4% PFA, embedded in paraffin and serially cut into 8 μm-thick sections. Some sections from each brain were also stained with cresyl violet to permit cytoarchitectural analysis. 2.4. Frozen sections method with postfixation Anesthetized animals were perfused with PBS only, and the brains were removed rapidly. Brain tissue was embedded in cryostat embedding medium in plastic mold and immediately frozen on top of liquid nitrogen until 70–80% of the block turned white. Coronal sections (10 μm) were cut in a cryostat at −18 °C and mounted onto poly-L-lysine coated slides. After drying at room temperature for

Polyclonal 1:2000

Iba1 (anti-ionized Polyclonal 1:1000 calcium binding adaptor molecule 1)

CD 4 cell surface antigen on T-helper lymphocytes, monocytes and macrophages CD8 antigen in a subset of T cells, thymocytes, and NK cells CD11a is leukocyte common antigen Integrin CD11b on macrophages and microglia Complement receptor 3 (CR3) on macrophages and microglia Integrin CD18 on macrophages and microglia Integrin αX chain on dendritic cells and some myeloid cells Membrane glycoprotein CD45 present on all leukocytes CD68 lysosomal membrane glycoprotein on macrophages Monomorphic determinant of MHC class II (β chain of RT1B) on activated microglia/macrophages, B lymphocytes, and dendritic cells Monomorphic determinant of MHC class II (RT1D) on activated microglia/ macrophages, B lymphocytes, and dendritic cells Integrin αE2 expressed by dendritic cells and T cells Phosphorylated tau protein (Ser-202, Thr-205) Phosphorylated tau protein (Thr-231) Tau protein Ionized calcium binding adaptor molecule 1 expressed by microglia and macrophages

Serotec

Serotec Serotec Serotec

Serotec Serotec

Serotec

Serotec

Serotec

Serotec

Serotec Pierce Pierce Axon Neuroscience WAKO

18

N. Zilka et al. / Journal of Neuroimmunology 209 (2009) 16–25

containing 0.3% Triton X-100 and 1% horse serum). Samples were subsequently incubated overnight at 4 °C in primary antibodies in blocking solution. After washing in PBS/0.3% Triton X-100, the sections were incubated with biotinylated secondary antibody (Vectastain; Vector Laboratories, Burlingame, CA). The reaction product was visualized using avidin–biotin and Vector DAB, Vector VIP or Vector SG as the chromogen (Vector Laboratories). Paraffin sections were counterstained with hematoxylin for 30 s. To investigate the extent of expression of different immunomarkers sensitive for fixation and paraffin embedding we used serial immunoperoxidasestained thin (10 μm) postfixed frozen sections.

rabbit polyclonal Iba 1 antibody. Both prefixed and postfixed sections were subsequently incubated with secondary antibodies conjugated with ALEXA 488 or ALEXA 546 fluorescent dyes (Invitrogen-Molecular Probes, Eugene, OR) for 1 h at room temperature. After washing, the sections were mounted onto slides using Vectashield mounting medium (Vector Laboratories), and examined with an Olympus IX 71 Fluoview laser scanning confocal microscope.

3. Results

2.7. Immunofluorescence

3.1. Activated microglia are distributed in the brain area affected by neurofibrillary degeneration in rats transgenic for human truncated tau protein

Co-localization experiments were performed on prefixed and postfixed frozen specimens. Postfixed sections were incubated for 8 h at 4 °C with the first primary antibodies (rabbit polyclonal antitau) followed by an overnight incubation (4 °C) with the second primary antibody (ED1 or ED8). Prefixed sections were incubated with a mixture of monoclonal anti-tau antibody (AT8 or AT180) and

The aim of this study was to analyze whether human truncated tau might induce inflammatory response and to identify the inflammatory pattern of this response. In order to detect and localize neurofibrillary degeneration and microglial activation, paraffin sagittal sections were prepared from control and transgenic rat brains. Neurofibrillary lesions were detected using antibody AT8, which recognizes tau

Fig. 1. Neurofibrillary pathology and activation of microglia in the gray matter of a transgenic rat expressing human truncated tau. In wild type SHR rats, immunostaining with the AT8 antibody (phosphotau Ser202, Thr205) does not reveal any presence of neurofibrillary pathology in the reticular formation of brainstem (A); monoclonal antibody ED-8 (CD11b/ CD18) recognizes only ramified resting microglia in the same area (D), while ED-1 (CD68) immunoreactivity is absent (G). In transgenic rats, the corresponding brain area is affected by massive neurofibrillary degeneration (B), accompanied by activated hypertrophic microglia (E) and with few macrophages (H); higher magnification shows neurofibrillary tangle, neuropil threads and dystrophic neurite (C), hypertrophic (white arrow) and clustering microglia (red arrow) (F) and CD68 immunopositive microglia (I). Paraffin sections. Scale bars: 100 μm (A, B, D, E, G, H), and 50 μm (C, F, I).

N. Zilka et al. / Journal of Neuroimmunology 209 (2009) 16–25

phosphorylated at Ser-202, Thr-205 (Braak et al., 1994; Goedert et al., 1995). Activated microglia were detected with monoclonal antibody ED8, which recognizes complement receptor 3 (CD11b/CD18) and with monoclonal antibody ED1, which recognizes lysosomal glycoprotein CD68. Our previous study showed that neurofibrillary lesions induced by in vivo expression of human truncated tau protein are distributed mainly in the brainstem and deep cerebellar nuclei of transgenic rats (Zilka et al., 2006; Koson et al., 2008). Three major neurofibrillary lesions were described in the vulnerable brain areas of transgenic rats: neurofibrillary tangles (NFTs), neuropil threads (NTs) and dystrophic neurites (Fig. 1B, C). Here we showed that activated microglia occurred in the same regions as AT8 immunoreactive tangles (Fig. 1E), but were conspicuously absent in areas with limited neurofibrillary degeneration such as thalamus, hypothalamus, striatum or cortex (data not shown). Furthermore, monoclonal antibody ED1 recognized characteristic cytoplasmic staining pattern in macrophages and in activated microglia cells (Fig. 1H). Two different morphologic types of activated microglia were identified: hypertrophic microglia with moderately enlarged soma and long, thick processes and clustering microglia (Fig. 1F). On the contrary, no neurofibrillary lesions were detected in age-matched control rat brains (Fig. 1A). In the brainstem nuclei and deep cerebellar nuclei, immunohistochemical staining with antibody ED8 revealed a staining pattern characteristic of resting microglia (Fig. 1D), while staining with ED1 antibody showed no immunoreactivity for CD68 in the same brain areas (Fig. 1G).

19

3.2. Immunophenotypic profile of inflammatory cells in the gray matter lesions The majority of NFT-bearing neurons stained with antibody AT180, which recognizes tau phosphorylated at Thr-231 (Goedert et al., 1994), were distributed throughout the brainstem nuclei (Fig. 2A). Neurofibrillary lesions were accompanied by a massive microglial reaction characterized by up-regulation of several immunomolecules including complement receptor 3 (Fig. 2B), CD4 (Fig. 2C) and CD45 (Fig. 2D). Histochemical analysis revealed perineuronal localization of several ED8-immunoreactive activated microglia cells. These cells had adopted a close relationship with the surface of large diameter brainstem neurons. Some of these perineuronally-localized activated microglia cells stained positive for CD4 and CD45 expression. Next we conducted an immunohistochemical analysis on brain sections from control and transgenic rat brains using monoclonal antibodies OX-6 and OX-17, which recognize, respectively, rat RT1B and RT1D antigens. This analysis revealed only a few OX-6- and OX-17-positive microglial cells which were detected in brains of transgenic rats (Fig. 2E, F). The figure shows the presence of only a small number of cells expressing RT1B and RT1D antigens. This finding, especially when compared to ED8 positive cells, suggests that MHCII-positive cells represent only a very small subpopulation of activated microglia. Age-matched controls showed no CD45 and MHC class II antigen labeling. Occasionally, CD4 immunopositive resting microglial cells were present in the white matter of non-transgenic rats.

Fig. 2. Upregulated immunomarkers on activated microglia and dendritic cells in the gray matter lesions. In transgenic rats, neurons in trigeminal nucleus are affected by neurofibrillary tangles and neuropil threads (A); the same brain area is infiltrated with activated microglia displaying up-regulated integrins CD11b/CD18 (B), lymphocytic antigen CD4 (C) and leukocyte common antigen CD45 (D). Only very limited number of RT1B (E) and RT1D (F) immunopositive cells are present in the lesions. Note the apposition of microglia around the neurons (B–C, small asterisks). Postfixed frozen sections. Scale bars: 100 μm.

20

N. Zilka et al. / Journal of Neuroimmunology 209 (2009) 16–25

Furthermore, we have analyzed the distribution of clustered microglia and their relationship to neurofibrillary tangles. We found that some of the phosphotau-positive neurofibrillary tangles immunolabeled with mAb AT8 colocalized with clustering microglia cells (Fig. 3A–C). Moreover, the clustering microglia expressed increased levels of lysosomal glycoprotein CD68 (Fig. 3D–F). 3.3. Activated microglia and macrophages are distributed within the white matter lesions in rats transgenic for human truncated tau protein In addition to neurofibrillary lesions, transgenic rats developed massive axonal degeneration mainly in the brainstem and spinal cord. In these areas, high numbers of dystrophic neurites and axonal spheroids were distributed within the white matter tracts (Fig. 4B, C). Interestingly, these pathological lesions are accompanied by extensive inflammatory responses manifested by ED1- and ED8-immunoreactive macrophages (Fig. 4E, F, H, I). Clustering of macrophages was a characteristic feature of their activation (Fig. 4F). These nodules typically contain several macrophages which appear to coalesce to form multinucleated immune cells. Examination of the age-matched control rats revealed no morphologically detectable pathological changes in the brainstem white matter tracts (Fig. 4A). Occasionally, a few ED1-expressing cells were identified in the same brain area (Fig. 4D). Sagittal sections from control SHR rats immunostained with ED8 antibody displayed a uniform staining pattern characteristic for resting microglia (Fig. 4G). 3.4. Immunophenotypic profile of inflammatory cells in the white matter lesions Immunohistochemical analysis was performed on coronal sections from rat brainstems to determine whether surface immunomarkers were overexpressed in white matter lesions of transgenic rats expressing human truncated tau. This analysis focused on antibodies against integrins CD11a, CD11b and CD18 and against immunomar-

kers CD4, CD45 and MHC class II antigens. We observed that axonal damage in the tectospinal tract (Fig. 5A) was accompanied by massive infiltration of macrophages forming large clusters. Macrophages displayed ameboid morphology with short surface processes suggesting their microglial origin. Immunohistochemical analysis revealed up-regulation of an array of immunomolecules including: CD11a (Fig. 5B), CD11b (Fig. 5C), CD18 (Fig. 5D), CD4 (Fig. 5E), CD45 (Fig. 5F). Furthermore, in the white matter lesions we failed to detect macrophages expressing the major histocompatibility complex class II antigen RT1B or RT1D. No axonal degeneration and inflammatory response was observed in age-matched controls. Moreover, confocal study showed that ED8 immunoreactive (Fig. 5G) and ED1-immunoreactive (Fig. 5H) macrophages were distributed in close proximity of tau-positive dystrophic neurites in the tectospinal tract. Frozen sections were fixed by acetone/ethanol which allowed us to selectively visualize assembled forms of tau protein, while soluble forms were washed out during the procedure. 3.5. Leukocyte infiltration of brain parenchyma In the affected brain areas of transgenic rats, clusters of inflammatory cells were detected in the intravascular, perivascular and paravascular space. The intravascular cell clusters and perivascular cell infiltrates were composed mainly of antigen-presenting cells with up-regulated RT1B antigen expression (Fig. 6A). As the inflammatory process progressed so the paravascular accumulations of leukocytes extended further out from the vessels (6B). Leukocytes were of uniform shape and size (typically round and approximately 10 μm in diameter). Their morphology and size made it easy to distinguish them from activated macrophages distributed throughout the area. Further characterization of these cells involved several monoclonal antibodies recognizing different populations of T lymphocytes (anti-CD4 for T-helper cells and anti-CD8 for cytotoxic T cells), antigen presenting cells (anti-RT1B and RT1D) and dendritic cells (anti-αE2 integrin, CD11c). An antibody specific for CD8

Fig. 3. Colocalization of clustered microglia and neurofibrillary tangles. The tangles were detected with monoclonal antibody AT8 recognizing tau protein phosphorylated at Ser-202, Thr-205 (A). Microglia were stained with polyclonal antibody Iba1, specific for ionized calcium binding adaptor molecule 1 (B, E). Confocal study showed that neurofibrillary tangles colocalized with clusters of activated microglia (C). Double immunostaining of CD68 (D) and Iba1 (E) shows co-expression of both antigens in clustered activated microglia (F). Prefixed frozen sections. Scale bars: 100 μm.

N. Zilka et al. / Journal of Neuroimmunology 209 (2009) 16–25

21

Fig. 4. Distribution of axonal lesions and activated microglia and macrophages in white matter tracts of transgenic rat. In age-matched control rats no axonal damage (A) and only few ED1 (CD68) (D) and ED8 (CD11b/CD18) (G) immunopositive glial cells are present in the white matter tracts of the brainstem. On the contrary, in the transgenic rats, massive axonal degeneration (AT8) is present (B, C); the same brain area shows ED1-immunopositive brain macrophages (E, F) and ED8-immunopositive activated microglia (H, I). Higher magnification shows dystrophic neurites with axonal swelling (C) and brain macrophages (F,I). Paraffin sections. Scale bars: 100 μm (A, B, D, E, G, H), and 50 μm (C, F, I).

receptors did not label brain parenchyma (data not shown), while CD4-immunoreactivity was mainly associated with activated microglia and macrophages. Further studies revealed that the infiltrating leukocytes expressed both RT1B and RT1D antigens (Fig. 6C, D) as well as the leukocyte common antigen CD45 (Fig. 6E). In addition, infiltrating leukocytes were immunostained for dendritic cell markers, CD11c and αE2 integrins (Matyszak and Perry, 1996; Sroga et al., 2003). We found that some of the blood borne leukocytes expressed integrin αE2 (Fig. 6F) but not CD11c. Finally, distribution of infiltrating leucocytes showed that they were preferentially attracted to the brain areas with neurofibrillary pathology. 4. Discussion We previously showed that non-mutated human truncated tau derived from sporadic Alzheimer's disease is able to induce and drive neurofibrillary degeneration in rats when expressed as transgene (Zilka et al., 2006; Koson et al., 2008). Transgenic rats displayed the AD-characteristic tau cascade consisting of tau hyperphosphorylation, formation of neurofibrillary tangles (NFT), sarcosyl-insoluble tau

complexes and axonal degeneration. These pathological changes led to the progressive decline of sensorimotor functions and impairment of several reflexes. This progressive neurobehavioral impairment reached a terminal stage characterized by pronounced neurological impairment, hunched posture, muscular weakness, bradykinesia and paraparesis (Hrnkova et al., 2007). In the present study we tested the hypothesis whether neurofibrillary lesions and axonal damage induced by human truncated tau protein may stimulate inflammatory response. The results described herein clearly show that in the transgenic rat brain, neurofibrillary lesions and axonal degeneration were closely associated with the distribution of reactive microglia and macrophages. We found that the type of lesion and its location were associated with differences in the relative distribution of microglia and macrophages. Differential involvement of activated microglia and macrophages was observed in the gray and white matter lesions. While activated microglia cells prevail in the gray matter lesions, macrophages were dominant in the white matter lesions. The finding that regional distribution of activated microglia parallels the distribution of phosphotau-positive neurofibrillary lesions suggests a similar inflammatory pattern of AD (Cras et al., 1991; Perlmutter et al., 1992;

22

N. Zilka et al. / Journal of Neuroimmunology 209 (2009) 16–25

Fig. 5. Upregulated immunomarkers on macrophages and activated microglia in the white matter lesions. Numerous dystrophic neurites are prominent in the tectospinal tract (A). Corresponding brain area is infiltrated by macrophages with upregulated integrins CD11a (B), CD11b (C) and CD18 (D), lymphocytic antigen CD4 (E) and leukocyte common antigen CD45 (F). Extensive degeneration of axonal fibers in the tectospinal tract (green) is surrounded by the ED8-immunopositive (G) and ED1-immunopositive (H) reactive microglia and macrophages (red). Postfixed frozen sections. Scale bars: 100 μm.

Dickson et al., 1996; DiPatre and Gelman, 1997; Oka et al., 1998; Overmyer et al., 1999; Sheffield et al., 2000) and non-AD tauopathies (Schwab et al., 1996; Ishizawa and Dickson, 2001; Imamura et al., 2001), where distribution of reactive microglia has been correlated with the presence of NFT. Microglia are resident macrophages forming the first line of defense and control the immune response in the brain. The striking feature of microglial cells is their rapid activation in response to even minor pathological changes in the CNS (Kreutzberg, 1996; Vilhardt, 2005). Activation of microglia displays a repertoire in terms of proliferation, migration to the site of injury, characteristic morphological, immunophenotypical (upregulation of innate immune cell surface receptors) and functional changes (antigen-presenting cell capabilities) (Streit et al., 1999). The distinctive morphological feature of the activated microglia is the development of enlarged cell processes, which give the cells a bushy appearance. Concomitant with cellular hypertrophy, activated microglia up-regulate a number

of immunomolecules including complement receptor 3 as well as lymphocytic antigen CD4 and leukocyte common antigen CD45 (Graeber et al., 1988, 1990; Stollg and Jander, 1999). In the presented transgenic rats expressing human truncated non-mutated tau protein, microglia undergo morphological changes from resting into the reactive state. Activation of microglia was accompanied by the upregulation of several surface immunomarkers including integrins CD11a, CD11b and CD18, lymphocytic antigen CD4, leukocyte common antigen CD45 and lysosomal glycoprotein CD68, indicating an inflammatory response similar to that seen in AD and related neurodegenerative disorders (Kobayashia et al., 1998; McGeer and McGeer, 1999). Another important issue that arises from our study regards the role of clustered microglia surrounding the neurofibrillary tangles. We showed that some of the NFTs expressing abnormally phosphorylated tau protein were “under the control” of reactive microglia and macrophages. Interestingly, these reactive immune cells showed

N. Zilka et al. / Journal of Neuroimmunology 209 (2009) 16–25

23

Fig. 6. Perivascular and paravascular infiltration of inflammatory leukocytes into brain parenchyma of transgenic rats. Numerous RT1B immunoreactive antigen presenting cells are cumulated in the blood vessel and in the perivascular space (A). Later, blood borne leukocytes migrate from perivascular space into injured brain parenchyma (B). Adjacent coronal frozen sections demonstrate perivascular cuffs of antigen presenting cells that are immunopositive for MHC class II antigens, namely RT1B (C) and RT1D (D) and for leukocyte common antigen CD45 (E). Some of these cells are immunopositive also for integrin αE2 (F), which is a marker for dendritic cells. Blood vessels are marked by arrows. Postfixed frozen sections. Scale bars: 100 μm (A, C, D, E, F), and 20 μm (B).

increased expression of the lysosomal glycoprotein CD68. It has been previously shown that CD68 plays a role in endocytosis or lysosomal traffic that is essential in antigen processing or presentation. These activities may have been expanded to become a dominant part of the specialized function of phagocytic cells such as macrophages where CD68 expression increases in the presence of phagocytic stimuli (Holness and Simmons, 1993; Holness et al., 1993; Ramprasad et al., 1996). Strikingly, in AD brains CD68-immunopositive microglia infiltrated extracellular NFTs and thus some authors hypothesized that microglia could be responsible for the modification of extracellular NFTs (Cras et al., 1991; Ikeda et al., 1992). On the basis of these findings we suggest that neurofibrillary structures in the brain of transgenic rats, which are surrounded by activated microglia/ macrophages with prominent lysosomal system, may represent extraneuronal “ghost” tangles. Interestingly, the inflammatory response in the white matter shows a different inflammatory pattern than in the gray matter. In the

area of axonal damage a high number of CD68-immunoreactive macrophages and CR3-immunoreactive clustering microglia were identified. It is difficult to identify the origin of the macrophages in the transgenic rat brain. It has been previously shown that, phenotypic differentiation of systemic macrophages that have infiltrated the central nervous system (CNS) and brain-derived macrophages still remains a serious problem in neurobiology (Guillemin and Brew, 2004). There is a lack of membranous and/or biochemical markers allowing conclusive identification of these cells. Strikingly, our study revealed that some of the macrophage like cells showed short processes on the cell surface suggesting their microglia origin. However, it is also possible that these cells were derived from both brain microglia and blood-borne leukocytes. In addition to the morphological and immunophenotypical changes, activation of microglia is defined by the functional differentiation characterized by upregulation of MHC class II antigens. In their quiescent state, microglia display low or undetectable levels of

24

N. Zilka et al. / Journal of Neuroimmunology 209 (2009) 16–25

MHC II class expression. However, after neuronal injury MHC class-II antigens can rapidly be upregulated in the entire population of resident microglia (Kreutzberg, 1996). One of the strongest stimuli for microglial cells to express MHC class II molecules is phagocytosis (Kösel et al., 1997). It was suggested that class II MHC antigens function as signal transducing molecules driving proinflammatory responses of microglia (Benveniste et al., 2001). Previous transgenic mice studies showed that deposition of mutated tau induced expression of MHC II antigens on the surface of activated microglia (Bellucci et al., 2004; Yoshiyama et al., 2007). Surprisingly, neuroinflammation induced by truncated tau was not accompanied by extensive up-regulation of MHC class II antigens. On the contrary, cells positive for RT1B and RT1D antigens, rat equivalent for human DQ and DR MHC class II molecules (Günther and Walter, 2001), represented only a small subpopulation of activated microglia. Interestingly, MHC class II antigen did not codistribute with macrophages present in the brainstem white matter affected by massive axonal degeneration. These results suggest that the expression of MHC class II molecules was not correlated with the severity of neurofibrillary degeneration or axonal damage. However, MHC class II immunoreactivity was observed on small perivascular cell clusters. These cells were morphologically distinct from microglia/ macrophages and are discussed below. It is important to mention that transgenic rats were produced on the genetic background of the SHR strain that has been characterized as immunodeficient rat strain (Takeichi et al., 1980). Previously it has been reported that transient middle cerebral artery occlusion led to a microglia and macrophage response showing apparent low levels of MHC class II antigen expression in the brain of SHR rats (Lehrmann et al., 1997). On the basis of our results we hypothesize that the apparent low presence of MHC class II positive cells may represent the strain-dependent differences in MHC class II expressions. The question whether blood-borne immune cells are infiltrating brain areas afflicted with neurodegeneration in AD and other tauopathies yielded contradictory results. Some authors claimed that the chronic inflammatory response implicated in neurodegenerative conditions was provided almost exclusively by resident CNS cells without any apparent influx of leukocytes from the blood (Eikelenboom et al., 2006; Streit et al., 2004). Others reported that hematopoietic cells can enter the brain in Alzheimer's disease and may contribute to an increased inflammatory burden (Itagaki et al., 1988; Togo et al., 2002; Fiala et al., 2002; Britschgi and Wyss-Coray, 2007; Ray et al., 2007). Recently, it has been shown in AD mice models that blood cells of the myeloid lineage infiltrate the brain, associate with the amyloid plaques and assume microglial properties (Malm et al., 2005; Stalder et al., 2005; Simard et al., 2006). Moreover, these cells were able to phagocytose β-amyloid and thus reduced the plaque burden in the brain (Glezer et al., 2006, Simard et al., 2006). However, the transgenic mice used in these studies underwent irradiation before bone marrow transplantation and this is known to result in vascular inflammation, activation and opening of the blood-brain barrier, increase of the level of inflammation by activation of glial cells and infiltration of leukocytes into the brain (Stalder et al., 2005; Chiang et al., 1993). In our transgenic rats, the innate immune response induced by human truncated tau promotes the influx of leukocytes from the circulation into the affected brain area in the absence of any irradiation. The infiltrating leukocytes carried surface markers of antigen presenting cells, including RT1B, RT1D and αE2 integrin, suggesting their monocyte/macrophage origin. These findings strongly suggest that antigen presenting cells (mainly monocytes) are able to enter the brain parenchyma afflicted by neurodegeneration. It remains to be evaluated how infiltrating leukocytes are contributing to the disease progression. Our study revealed that human misfolded truncated tau protein is able to promote the inflammatory response manifested by a significant upregulation of immune-molecules and by activation of microglia and macrophages with influx of antigen-presenting cells

from blood into the brain parenchyma of transgenic rats. These observations emphasize the potential value of transgenic rats as a proper animal model for the study of a) the sequence of events in the neurodegeneration driven inflammatory response at the transcriptomic and proteomic level focused on gene expression and protein profile studies of proteins of complement cascades, Toll like receptors, cytokines and chemokines; b) the causal relationships between genetic background and inflammatory response in the brain parenchyma — using various rat strains and c) the link between cerebrospinal immunoproteome and disease progression and translate it into a potential diagnostic, prognostic and therapeutic use. Moreover, understanding the molecular mechanisms that modulate neuroinflammatory responses and their impact on neurodegenerative processes would lead to the identification of novel inflammatory pathways involved in the tau cascade and to the development of inflammation-based therapeutic strategies for human tauopathies. Acknowledgement This work was supported by research grants APVV-0631-07, LPP0326-06, LPP-0353-06, LPP-0354-06 and LPP-0363-06. The authors wish to thank Assoc. Prof. Colin Campbell, PhD., University of Minnesota, USA, for advice and careful reading of the manuscript. References Aitman, T.J., Glazier, A.M., Wallace, C.A., Cooper, L.D., Norsworthy, P.J., Wahid, F.N., Al-Majali, K.M., Trembling, P.M., Mann, C.J., Shoulders, C.C., Graf, D., St. Lezin, E., Kurtz, T.W., Kren, V., Pravenec, M., Ibrahimi, A., Abumrad, N.A., Stanton, L.W., Scott, J.C., 1999. Identification of Cd36 (Fat) as an insulin resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats. Nat. Gen. 21, 76–83. Akiyama, H., Arai, T., Kondo, H., Tanno, E., Haga, C., Ikeda, K., 2000. Cell mediators of inflammation in the Alzheimer disease brain. Alzheimer Dis. Assoc. Disord. 14, 47–53. Bellucci, A., Westwood, A.J., Ingram, E., Casamenti, F., Goedert, M., Spillantini, M.G., 2004. Induction of inflammatory mediators and microglial activation in mice transgenic for mutant human P301S tau protein. Am. J. Pathol. 165, 1643–1652. Benveniste, E.N., Nguyen, V.T., O'Keefe, G.M., 2001. Immunological aspects of microglia: relevance to Alzheimer's disease. Neurochem. Int. 39, 381–391. Blasko, I., Grubeck-Loebenstein, B., 2003. Role of the immune system in the pathogenesis, prevention and treatment of Alzheimer's disease. Drugs Aging 20, 101–113. Blasko, I., Stampfer-Kountchev, M., Robatscher, P., Veerhuis, R., Eikelenboom, P., Grubeck-Loebenstein, B., 2004. How chronic inflammation can affect the brain and support the development of Alzheimer's disease in old age: the role of microglia and astrocytes. Aging Cell 3, 169–176. Braak, E., Braak, H., Mandelkow, E.M., 1994. A sequence of cytoskeleton changes related to the formation of neurofibrillary tangles and neuropil threads. Acta Neuropathol. 87, 554–567. Britschgi, M., Wyss-Coray, T., 2007. Systemic and acquired immune responses in Alzheimer's disease. Int. Rev. Neurobiol. 82, 205–233. Chiang, C.S., McBride, W.H., Withers, H.R., 1993. Radiation-induced astrocytic and microglial responses in mouse brain. Radiother. Oncol. 29, 60–68. Cras, P., Kawai, M., Siedlak, S., Perry, G., 1991. Microglia are associated with the extracellular neurofibrillary tangles of Alzheimer disease. Brain Res. 558, 312–314. D'Andrea, M.R., Cole, G.M., Ard, M.D., 2004. The microglial phagocytic role with specific plaque types in the Alzheimer disease brain. Neurobiol. Aging 25, 675–683. Dickson, D.W., Sinicropi, S., Yen, S.H., Ko, L.W., Mattiace, L.A., Bucala, R., Vlassara, H., 1996. Glycation and microglial reaction in lesions of Alzheimer's disease. Neurobiol. Aging 17, 733–743. DiPatre, P.L., Gelman, B.B., 1997. Microglial cell activation in aging and Alzheimer disease: partial linkage with neurofibrillary tangle burden in the hippocampus. J. Neuropathol. Exp. Neurol. 56, 143–149. Eikelenboom, P., Veerhuis, R., Scheper, W., Rozemuller, A.J.M., van Gool, W.A., Hoozemans, J.J.M., 2006. The significance of neuroinflammation in understanding Alzheimer's disease. J. Neural Transm. 113, 1685–1695. Fiala, M., Liu, Q.N., Sayre, J., Pop, V., Brahmandam, V., Graves, M.C., Vinters, H.V., 2002. Cyclooxygenase-2-positive macrophages infiltrate the Alzheimer's disease brain and damage the blood-brain barrier. Eur. J. Clin. Invest. 32, 360–371. Glezer, I., Simard, A.R., Rivest, S., 2006. Neuroprotective role of the innate immune system by microglia. Neurosci. 147, 867–883. Goedert, M., Jakes, R., Crowther, R.A., Cohen, P., Vanmechelen, E., Vandermeeren, M., Cras, P., 1994. Epitope mapping of monoclonal antibodies to the paired helical filaments of Alzheimer's disease: identification of phosphorylation sites in tau protein. Biochem. J. 301, 871–877. Goedert, M., Jakes, R., Vanmechelen, E., 1995. Monoclonal antibody AT8 recognises tau protein phosphorylated at both serine 202 and threonine 205. Neurosci Lett. 189, 167–170 (4).

N. Zilka et al. / Journal of Neuroimmunology 209 (2009) 16–25 Graeber, M.B., Streit, W.J., Kiefer, R., Schoen, S.W., Kreutzberg, G.W., 1990. New expression of myelomonocytic antigens by microglia and perivascular cells following lethal motor neuron injury. J. Neuroimmunol. 27, 121–132. Graeber, M.B., Streit, W.J., Kreutzberg, G.W., 1988. Axotomy of the rat facial nerve leads to increased CR3 complement receptor expression by activated microglial cells. J. Neurosci. Res. 21, 18–24. Guillemin, G.J., Brew, B.J., 2004. Microglia, macrophages, perivascular macrophages, and pericytes: a review of function and identification. J. Leukoc. Biol. 75, 388–397. Günther, E., Walter, L., 2001. The major histocompatibility complex of the rat (Rattus norvegicus). Immunogenetics 53, 520–542. Holness, C., da Silva, R.P., Fawcett, J., Gordon, S., Simmons, D.L., 1993. Macrosialin, a mouse macrophage-restricted glycoprotein, is a member of the lamp/lgp family. J. Biol. Chem. 268, 9661–9666. Holness, C.L., Simmons, D.L., 1993. Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins. Blood 81, 1607–1613. Hrnkova, M., Zilka, N., Minichova, Z., Koson, P., Novak, M., 2007. Neurodegeneration caused by expression of human truncated tau leads to progressive neurobehavioural impairment in transgenic rats. Brain Res. 1130, 206–213. Ikeda, K., Akiyama, H., Haga, C., Haga, S., 1992. Evidence that neurofibrillary tangles undergo glial modification. Acta Neuropathol. 85, 101–104. Imamura, K., Sawada, M., Ozaki, N., Naito, H., Iwata, N., Ishihara, R., Takeuchi, T., Shibayama, H., 2001. Activation mechanism of brain microglia in patients with diffuse neurofibrillary tangles with calcification: a comparison with Alzheimer disease. Alzheimer Dis. Assoc. Disord. 15, 45–50. Ishizawa, K., Dickson, D.W., 2001. Microglial activation parallels system degeneration in progressive supranuclear palsy and corticobasal degeneration. J. Neuropathol. Exp. Neurol. 60, 647–657. Itagaki, S., McGeer, P.L., Akiyama, H., 1988. Presence of T-cytotoxic supressor and leukocyte common antigen positive cells in Alzheimer's disease brain tissue. Neurosci. Lett. 91, 259–264. Kobayashia, K., Muramoria, F., Aokia, T., Hayashib, M., Miyazub, K., Fukutanic, Y., Mukaic, M., Koshinoa, F.,1998. KP-1 is a marker for extraneuronal neurofibrillary tangles and senile plaques in Alzheimer diseased brains. Dement. Geriat. Cog. Dis. 9, 13–19. Kösel, S., Egensperger, R., Bise, K., Arbogast, S., Mehraein, P., Graeber, M.B., 1997. Longlasting perivascular accumulation of major histocompatibility complex class IIpositive lipophages in the spinal cord of stroke patients: possible relevance for the immune privilege of the brain. Acta Neuropathol. 94, 532–538. Koson, P., Zilka, N., Kovac, A., Kovacech, B., Korenova, M., Filipcik, P., Novak, M., 2008. Truncated tau expression levels determine life span of rat model of tauopathy but not a neuronal loss and the terminal load of neurofibrillary tangles. Eur. J. Neurosci. 28 (2), 239–246. Kreutzberg, G.W., 1996. Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19, 312–318. Lehrmann, E., Christensen, T., Zimmer, J., Diemer, N.H., Finsen, B., 1997. Microglial and macrophage reactions mark progressive changes and define the penumbra in the rat neocortex and striatum after transient middle cerebral artery occlusion. J. Comp. Neurol. 386, 461–476. Lukiw, W.J., Bazan, N.G., 2000. Neuroinflammatory signaling upregulation in Alzheimer's disease. Neurochem. Res. 25, 1173–1184. Malm, T.M., Koistinaho, M., Parepalo, M., Vatanen, T., Ooka, A., Karlsson, S., Koistinaho, J., 2005. Bone-marrow-derived cells contribute to the recruitment of microglial cells in response to beta-amyloid deposition in APP/PS1 double transgenic Alzheimer mice. Neurobiol. Dis. 18, 134–142. Matyszak, M.K., Perry, V.H., 1996. The potential role of dendritic cells in immunemediated inflammatory diseases in the central nervous system. Neuroscience 74, 599–608. McGeer, P.L., McGeer, E.G., 1995. The inflammatory response system of brain: implications for therapy of Alzheimer and other neurodegenerative diseases. Brain Res. Brain Res. Rev. 21, 195–218. McGeer, P.L., McGeer, E.G., 1999. Inflammation of the brain in Alzheimer's disease: implications for therapy. J. Leukoc. Biol. 65, 409–415. McGeer, P.L., McGeer, E.G., 2002. Local neuroinflammation and the progression of Alzheimer's disease. J. Neurovirol. 8, 529–538. McGeer, P.L., McGeer, E.G., 2004. Inflammation and the degenerative diseases of aging. Ann. N. Y. Acad. Sci. 1035, 104–116. Mrak, R., Griffin, W.S.T., 2005. Glia and their cytokines in progression of neurodegeneration. Neurobiol. Aging 26, 349–354.

25

Mrak, R.E., Griffin, W.S., 2001. The role of activated astrocytes and of the neurotrophic cytokine S100B in the pathogenesis of Alzheimer's disease. Neurobiol. Aging 22, 915–922. Oka, M., Katayama, S., Watanabe, C., Noda, K., Mao, J.J., Nakamura, S., 1998. Argyrophilic structures stimulate glial reactions in neurofibrillary tangles and senile plaques. Neurol. Res. 20, 121–126. Overmyer, M., Helisalmi, S., Soininen, H., Laakso, M., Riekkinen, P., Alafuzoff, I., 1999. Reactive microglia in aging and dementia: an immunohistochemical study of postmortem human brain tissue. Acta Neuropathol, 97, 383–392. Perlmutter, L.S., Scott, S.A., Barron, E., Chui, H.C., 1992. MHC class II-positive microglia in human brain: association with Alzheimer lesions. J. Neurosci. Res. 33, 549–558. Ramprasad, M., Terpstra, V., Kondratenko, N., Quehenberger, O., Steinberg, D., 1996. Cell surface expression of mouse macrosialin and human CD68 and their role as macrophage receptors for oxidized low density lipoprotein. Proc. Natl. Acad. Sci. U.S.A. 93, 14833–14838. Ray, R., Britschgi, M., Herbert, C., Takeda-Uchimura, Y., Boxer, A., Blennow, K., Friedman, L.F., Galasko, D.R., Jutel, M., Karydas, A., Kaye, J.A., Leszek, J., Miller, B.L., Minthon, L., Quinn, J.F., Rabinovici, G.D., Robinson, W.H., Sabbagh, M.N., So, Y.T., Sparks, D.L., Tabaton, M., Tinklenberg, J., Yesavage, J.A., Tibshirani, R., Wyss-Coray, T., 2007. Classification and prediction of clinical Alzheimer's diagnosis based on plasma signaling proteins. Nat. Med. 82, 1–4. Rogers, J., Strohmeyer, R., Kovelowski, C.J., Li, R., 2002. Microglia and inflammatory mechanisms in the clearance of amyloid β peptide. Glia 40, 260–269. Schwab, C., Steele, J.C., McGeer, P.L., 1996. Neurofibrillary tangles of Guam Parkinsondementia are associated with reactive microglia and complement proteins. Brain Res. 707, 196–205. Sheffield, L.G., Marquis, J.G., Berman, N.E., 2000. Regional distribution of cortical microglia parallels that of neurofibrillary tangles in Alzheimer's disease. Neurosci. Lett. 285, 165–168. Sheng, J.G., Mrak, R.E., Griffin, W.S., 1997. Glial-neuronal interactions in Alzheimer disease: progressive association of IL-1alpha+ microglia and S100beta+ astrocytes with neurofibrillary tangle stages. J. Neuropathol. Exp. Neurol. 56, 285–290. Simard, A.R., Soulet, D., Gowing, D., Julien, J.P., Rivest, S., 2006. Bone marrow derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease. Neuron 48, 489–502. Sroga, M.J., Jones, T.B., Kigerl, K.A., Mcgaughy, V.M., Popovich, P.G., 2003. Rats and mice exhibit distinct inflammatory reactions after spinal cord injury. J. Comp. Neurol. 462, 223–240. Stalder, A.K., Ermini, F., Bondolfi, L., Krenger, W., Burbach, G.J., Deller, T., Coomaraswamy, J., Staufenbiel, M., Landmann, R., Jucker, M., 2005. Invasion of hematopoietic cells into the brain of amyloid precursor protein transgenic mice. J. Neurosci. 25, 11125–11132. Stollg, G., Jander, S., 1999. The role of microglia and macrophages in the pathophysiology of the CNS. Prog. Neurobiol. 58, 233–247. Streit, W.J., Mrak, R., Griffin, W.S.T., 2004. Microglia and neuroinflammation: a pathological perspective. J. Neuroinflamm. 14, 1–4. Streit, W.J., Walter, S.A., Pennell, N.A., 1999. Reactive Microgliosis. Progress Neurobiol. 57, 563–581. Takeichi, N., Suzuki, K., Okayasu, T., Kobayashi, H., 1980. Immunological depression in spontaneously hypertensive rats. Clin. Exp. Immunol. 40, 120–126. Togo, T., Akiyama, H., Iseki, E., Kondo, H., Ikeda, K., Kato, M., Oda, T., Tsuchiya, K., Kosaka, K., 2002. Occurrence of T cells in the brain of Alzheimer's disease and other neurological diseases. J. Neuroimmunol. 124, 83–92. Tuppo, E.E., Arias, H.R., 2005. The role of inflammation in Alzheimer's disease. Int. J. Biochem. Cell Biol. 37, 289–305. Vilhardt, F., 2005. Microglia: phagocyte and glia cell. Int. J. Bioch. Cell Biol. 37, 17–21. Walsch, S., Aisen, P., 2004. Inflammatory processes in Alzheimer's disease. Expert Rev. Neurotherapeutics 4, 793–798. Wyss-Coray, T., Mucke, L., 2002. Inflammation in neurodegenerative disease — a double-edged sword. Neuron 35, 419–432. Yoshiyama, Y., Higuchi, M., Zhang, B., Huang, S.M., Iwata, N., Saido, T.C., Maeda, J., Suhara, T., Trojanowski, J.Q., Lee, V.M., 2007. Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model. Neuron 53, 337–351. Zilka, N., Filipcik, P., Koson, P., Fialova, L., Skrabana, R., Zilkova, M., Rolkova, G., Kontsekova, E., Novak, M., 2006. Truncated tau from sporadic Alzheimer's disease suffices to drive neurofibrillary degeneration in vivo. FEBS Lett. 580, 3582–3588.