J Neural Transm (2011) 118:737–745 DOI 10.1007/s00702-011-0626-5
BASIC NEUROSCIENCES, GENETICS AND IMMUNOLOGY - ORIGINAL ARTICLE
Reduced secretagogin expression in the hippocampus of P301L tau transgenic mice Johannes Attems • Arne Ittner • Kurt Jellinger • Roger M. Nitsch • Magdalena Maj • Ludwig Wagner • Ju¨rgen Go¨tz • Mathias Heikenwalder
Received: 2 October 2010 / Accepted: 13 March 2011 / Published online: 26 March 2011 Ó Springer-Verlag 2011
Abstract Neuropathological features in Alzheimer’s Disease (AD) include the presence of hyperphosphorylated forms of the microtubule-associated tau protein (tau) in hippocampal neurones. Numerous studies indicate a neuroprotective effect of calcium-binding proteins (Ca2? binding proteins) in neurodegenerative diseases (e.g., AD). Secretagogin is a newly described Ca2? binding protein that is produced by pyramidal neurones of the human hippocampus. Recently, secretagogin expressing hippocampal
J. Attems (&) Institute for Ageing and Health, Wolfson Research Centre, Newcastle University, NE4 5PL Newcastle upon Tyne, UK e-mail:
[email protected] A. Ittner J. Go¨tz Alzheimer’s and Parkinson’s Disease Laboratory, Brain and Mind Research Institute, The University of Sydney, 100 Mallett Street, Camperdown, NSW 2050, Australia K. Jellinger Institute for Clinical Neurobiology, Kenyongasse 18, 1070 Vienna, Austria R. M. Nitsch Division for Psychiatry Research, University Hospital Zu¨rich, August Forel Strasse 1, 8008 Zu¨rich, Switzerland M. Maj L. Wagner Departement of Medicine III, Medical University of Vienna, Wa¨hringer Gu¨rtel 18-20, 1090 Vienna, Austria M. Heikenwalder Institute for Virology, Technische Universita¨t Mu¨nchen (TUM), Helmholtz Zentrum Mu¨nchen (HMGU) Schneckenburgstrasse 8, 81675 Mu¨nchen, Germany M. Heikenwalder Institute for Neuropathology, University Hospital Zu¨rich, Schmelzbergstrasse 12, 8091 Zu¨rich, Switzerland
neurones were demonstrated to resist tau-induced pathology in AD in contrast to the majority of neighbouring neurones. This suggested a neuroprotective effect of secretagogin in hippocampal neurones. Here, we investigated secretagogin expression in wild type (wt) mice as well as in hemizygous and homozygous P301L tau transgenic (tg) mice, which show pronounced and widespread tau pathology in hippocampal neurones. Secretagogin expression was analyzed at the immunohistochemical and biochemical levels in brains of age-matched wt and hemi- and homozygous tau tg mice. In wt mice hippocampal secretagogin-immunoreactive neurones were invariably detected, while immunoreactivity was much lower (P \ 0.001) in tau tg mice. Of note, hippocampal secretagogin immunoreactivity was absent in 62.5% of homozygous tau tg mice. In line with this finding, Western blot analysis demonstrated a significant reduction in protein expression levels of secretagogin in homozygous tau tg compared to wt mice. Our results suggest that increased levels of tau negatively influence secretagogin expression in the hippocampus of tau tg mice. Keywords Alzheimer’s disease Calcium-binding protein Secretagogin P301L tau transgenic mouse Tau pathology
Introduction Neuropathologically, Alzheimer’s disease (AD) is characterized by the presence of both amyloid-b (Ab) depositions in the form of plaques and of hyperphosphorylated microtubule-associated tau-protein (tau) in the form of neuropil threads (NT) and neurofibrillary tangles (NFT) (Braak et al. 2006; Hyman 1998; Mirra et al. 1993). The presence of NTs and NFTs is collectively referred to as tau
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pathology. These neuropathological features are partly attributed to alterations in the function of calcium-binding proteins (Ca2? binding proteins) (Abu-Soud and Stuehr 1993; Drewes et al. 1993; Gandhi and Keenan 1983; Solomon et al. 2001; Thibault et al. 1998). EF-hand proteins are a family of Ca2? binding proteins, which share a unique tandem repeat of the calcium-binding loop flanked by two a-helices known as the ‘‘EF-hand’’ calcium-binding site (Celio et al. 1996). With respect to their function, two groups of EF-hand proteins have been identified and classified so far: (1) The first group comprises of Ca2? binding proteins that undergo substantial conformational changes upon calcium-binding (Burgoyne and Weiss 2001; Dalgarno et al. 1984) thus acting as ‘‘triggers’’ and starting a cascade of reactions. This includes the calcium sensor proteins calmodulin and calmyrin. In the central nervous system (CNS), they have specific functions, such as the calcium sensor proteins in the retina, or broader functions in neurotransmitter release, channel and receptor regulation, control of gene transcription, neuronal growth and survival (Burgoyne 2007). The proteins in this group bind calcium with an affinity above resting free calcium concentration (Ikura 1996). (2) The second group of Ca2? binding proteins acts as ‘‘buffers’’, by decreasing free cytoplasmic calcium concentrations. They bind calcium with high affinity but do not undergo significant conformational changes. Parvalbumin, calbindin and calretinin belong to this group (Braunewell and Gundelfinger 1999; Hof et al. 1999; Ikura 1996; Skelton et al. 1994). The role of Ca2? binding proteins in neurodegenerative diseases (e.g., AD) remains elusive and the exact molecular and cellular pathways involved in protective as well as detrimental functions of Ca2? binding proteins and what determines selective neuronal vulnerability are unknown (Gotz et al. 2009). However, numerous studies have so far indicated a neuroprotective effect of Ca2? binding proteins (Blandini et al. 2004; Greene et al. 2001; Hof et al. 1993; Iacopino and Christakos 1990; Leuba et al. 1998). For example, neurones containing parvalbumin appear to be resistant to ischemia largely through their ability to buffer the rise of intracellular calcium (Arai et al. 1987; Ferrer et al. 1991; Fonseca et al. 1993; Hof et al. 1991; Inaguma et al. 1992; Leifer and Kowall 1993; Nitsch et al. 1989; Satoh et al. 1991; Scharfman and Schwartzkroin 1989). At present, however, it is unclear whether parvalbumin immunoreactive neurones in the cortex are resistant to neurodegeneration (Ferrer et al. 1991; Fonseca et al. 1993; Hof et al. 1991) or whether they undergo pathological changes typical of AD (Arai et al. 1987; Inaguma et al. 1992; Satoh et al. 1991). Secretagogin is an EF-hand Ca2? binding protein (Wagner et al. 2000) that contains six EF-hand calcium-
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binding domains and has a molecular weight of 32 kDa. In humans, its gene is located on chromosome 6p22 [GenBankTM accession number Y16752, for details see (Wagner et al. 2000)]. Secretagogin immunoreactivity and expression were detected in the gastrointestinal tract, thyroid, adrenal medulla, and in the brain (Attems et al. 2007; Gartner et al. 2001; Maj et al. 2010). In the CNS secretagogin was shown to interact with SNAP-25 (25 kDa synaptosome-associated protein), a protein that is involved in calcium-induced neurotransmitter exocytosis as well as in memory consolidation and long-term memory formation in the hippocampus (Hou et al. 2004; Hou et al. 2006; Rogstam et al. 2007). We recently demonstrated that secretagogin immunoreactivity in the human hippocampus is strictly confined to pyramidal neurones showing a hippocampal region specific distribution (i.e., CA3[CA2[CA4[CA1), which is not influenced by age, gender, or AD pathology (e.g., Ab and tau) (Attems et al. 2007). Moreover, post-mortem brains of AD cases show secretagogin expressing neurones in the hippocampus that did not succumb to tau pathology. The latter is indicated by the virtual absence of a co-localization between secretagogin and tau. In addition, Western blot analysis does not reveal differences in secretagogin protein expression between AD cases and controls (Attems et al. 2008). Therefore, it was hypothesized that secretagogin expressing neurones are largely resistant to tau-associated neurodegeneration in AD. In order to further elucidate the presumed neuroprotective function of secretagogin we sought to evaluate secretagogin immunoreactivity and expression in the hippocampus of wild type (wt) mice and both, hemi- and homozygous (P301L) tau transgenic (tg) mice with a pronounced tau pathology in cortex, amygdala and hippocampus (Deters et al. 2008; Gotz et al. 2001). These tau tg mice present with tau pathology in the absence of Ab plaques.
Materials and methods Mice Mice were kept under specified pathogen free (SPF) conditions in accordance with the legislation of the Veterinarian Office of the Kanton Zu¨rich. For immunohistochemical analysis, we investigated post-mortem brains from 15 wt (C57BL/6; 8 female and 7 male), 8 hemizygous (8 female) and 16 homozygous (13 female and 3 male) tau tg (P301L) mice. These mice express the longest isoform of human tau, together with the pathogenic mutation P301L, under the control of the mThy1.2 promoter (Gotz et al. 2001). The strain has been back-crossed onto C57BL/6 as
Reduced secretagogin expression in the hippocampus of mice
described (Pennanen et al. 2004). The mean age of mice was 10.24 months (range, 6–13; ±SE, 0.27), 11 months for wt (range, 6–12; ±SE, 0.48), 9.14 months for hemizygous (range 9–10; ±SE, 0.13) and 10 months for homozygous (range, 7–13; ±SE, 0.4) tau tg mice. Immunohistochemistry To detect secretagogin expression in the hippocampus sections of all murine brains investigated in this study were incubated with a polyclonal rabbit anti-secretagogin antibody. In order to assess overall secretagogin immunoreactivity step-cuts with 500 lm intervals in both frontal and para-sagittal planes from a subset of 3 wt and 3 homozygous tau tg mice brains were additionally incubated with the same antibody. The specificity of the antibody has been confirmed previously (Wagner et al. 2000) and the antibody has since been used in several studies (Attems et al. 2007; Birkenkamp-Demtroder et al. 2005; Gartner et al. 2001; Rogstam et al. 2007; Zhan et al. 2003; Zierhut et al. 2005). Brain tissue was fixed in 4% formaldehyde (PH7 buffered) for at least 2 weeks. Paraffinembedded tissue sections containing the hippocampus were incubated with polyclonal rabbit anti-secretagogin antibody as described previously (Gartner et al. 2001; Wagner et al. 2000): Paraffin-embedded tissue sections were deparaffinized and rehydrated by sequential incubations in xylene and a graded alcohol series (100, 96, and 80%). Prior to immunohistochemical analysis, tissue sections were regenerated in citrate-buffer PH6 (DAKO, Glostrup, Denmark) in a microwave oven (3 min, 630 watt and 30 min, 240 watt). Tissue sections were incubated with the polyclonal rabbit anti-secretagogin antibody (diluted 1:3,000) for 32 min at room temperature and stained sequentially with polyclonal swine anti-rabbit antibody (DAKO, Glostrup, Denmark), followed by Streptavidin– Biotin–Alkaline–Phosphatase complex (DAKO, Glostrup, Denmark), both diluted in 1:100 PBS containing BSA (10 ng/ll), and then developed with Fast Red (ZYMED, San Francisco, CA) as chromogenic substrate. Slides were counterstained with haemalaun and mounted with Glycergel (DAKO, Glostrup, Denmark). All incubations were performed in a moist chamber at room temperature. Between each incubation step, the slides were washed twice for 5 min with Tris puffer containing 0.1% Tween 20. It was shown previously, that homozygous tau tg mice express high levels of tau in the hippocampus (Gotz et al. 2001). We evaluated tau immunoreactivity in all tau tg mice (hemizygous and homozygous) and in 3 wt mice in sections adjacent to the ones which were used for secretagogin immunohistochemistry. Tau immunohistochemistry was performed using the AT8 antibody according to the manufacturer’s directions (Innogenetics, Ghent, Belgium).
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Double immunofluorescence Fixed brains of 3 homozygous tau tg mice were embedded in paraffin using an Excalibur tissue processor (Thermo). Sections (3 lm) were subjected to antigen retrieval in a temperature- and pressure-controlled microwave system (Milestone) in citrate buffer (10 mM pH 5.8) for 1 min at 120°C, followed by cooling under running tap water for 10 min. Primary antibodies were human tau-specific Tau13 (Tau13; Abcam) and Secretagogin. Alexa-coupled secondary antibodies were used for detection. Western blotting Hippocampi were isolated from 3 homozygous tau tg and 3 wt littermate mice (age 6 months). Tissue was lysed in RIPA buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1% NP40, 5 mM EDTA, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) by sonication and centrifuged at 20,000 g for 10 min at 4°C. Equal amounts of protein sample (150 lg) were separated by SDS PAGE and transferred onto nitrocellulose. Polyclonal rabbit antiSecretagogin antibody was used at 1:1,000, anti-GAPDH was used at 1:5,000. Quantification was performed by densitometric measurement using the Image J software. Evaluation and statistics In mid-frontal sections containing the second third (anterior– posterior) of the hippocampus secretagogin immunoreactivity in the hippocampus, including CA1–CA3 and dentate gyrus, was scored semiquantitatively using a four tiered scoring system: no neuropil immunoreactivity and no positive neurones (score 0, e.g., Fig. 1c), some sparse neuropil immunoreactivity and/or some single positive neurones (score 1), moderate neuropil immunoreactivity and up to 50 positive neurones (score 2, e.g., Fig. 1e), widespread and strong neuropil immunoreactivity and over 50 positive neurones in the hippocampus (score 3, e.g., Fig. 1a, b). In adjacent sections, tau immunoreactivity was evaluated semiquantitatively according to standardized criteria (Alafuzoff et al. 2008): absent (0), low (1), moderate (2) and high (3). Except for quantitative assessment of Western blot data (Student’s t test), our data were not normally distributed (Kolmogorov–Smirnov P \ 0.05); hence we used nonparametric tests for statistical analysis. Image acquisition Images of immunohistochemically stained sections were taken using a Nikon 90i microscope equipped with a digital camera head (Nikon, DS-Qi1Mc) coupled to a PC with Nikon NIS Elements imaging software. For double
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Fig. 1 Secretagogin in the hippocampus of wt mice and tg tau mice (P301L) with tau pathology. (a and b) Secretagogin immunoreactivity (score 3) in wt mice in the neuropil of stratum oriens (a; empty arrowheads in b), stratum lacunosum moleculare (a; asterisks in b, b1 and b2; b1, blue frame in b; b2, green frame in b) and the molecular layer of the dentate gyrus (a; full arrowheads in b and b2) while cellular immunoreactivity is present granule cell layer of the dentate gyrus (a; dots in b; arrows in b2) and in hippocampal pyramidal cells (arrows in b1). The cortex in wt mice shows no secretagogin immunoreactivity (a). (c) No secretagogin immunoreactivity (score 0) in both cortex and hippocampus of homozygous tau tg mice that show abundant tau pathology in the cortex (d; arrows in d1; d1, blue frame
in d) and in both granule cell layer (d) and stratum lacunosum moleculare (d; arrows in d2; d2, green frame in d) of the dentate gyrus (c and d are adjacent sections). (e) A hemizygous tau tg mouse shows secretagogin immunoreactivity (score 2) in both stratum moleculare and granule cell layer of dentate gyrus. scgn in lower right corner indicates staining with polyclonal secretagogin antibody and AT8 with tau antibody (for details see ‘‘Immunohistochemistry’’). Ctx cortex, so stratum oriens, sp stratum pyramidale, sr stratum radiatum, slm stratum lacunosum moleculare, gr granule cell layer, DG dentate gyrus, CA1 cornu ammonis sector 1, CA2 cornu ammonis sector 2, CA3 cornu ammonis sector 3. Scalebars, 200 lm
immunofluorescence, imaging was performed on an Olympus BX51 epifluorescence microscope.
were detected in all wt mice. Here, immunoreactivity was homogenously distributed throughout the soma as well as axons predominantly in neurones of sectors CA2 and CA3 and in the granule cell layer of the dentate gyrus (Fig. 1a, b), while the cortex did not show any secretagogin immunoreactive neurones. Neuropil immunoreactivity was present in stratum oriens, stratum lacunosum molecular and the molecular layer of the dentate gyrus (Fig. 1a, b). Of 15 wt
Results We first analyzed expression of secretagogin in hippocampi of wt mice. Secretagogin immunoreactive neurones
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cases, 9 showed secretagogin immunoreactivity fulfilling the criteria for score 3 and 5 wt cases showed moderate immunoreactivity consistent with score 2. In 1 wt case only sparse immunoreactivity could be detected (score 1). The mean secretagogin score in wt mice was 2.53 (median, 3; SE, ±0.17, Fig. 2). As expected, no tau immunoreactivity was observed in wt mice. We further assessed secretagogin immunoreactive neurones in tau tg mice: expression of secretagogin in the hippocampus was observed in 4 out of 8 hemizygous tau tg mice; one case fulfilled the criteria for score 2 (Fig. 1e) while in the remaining 3 cases only sparse immunoreactivity
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was observed (score 1; mean, 0.63; median, 1; SE, ±0.26; Table 1). Immunoreactivity for tau in hippocampal neurones was observed in 3 out of 8 hemizygous tau tg mice. Of note, all tau positive hemizygous tau tg mice lacked immunoreactivity for secretagogin in the hippocampus (score 0, Table 1). Additionally, we analyzed immunoreactivity for secretagogin in homozygous tau tg mice. Immunoreactivity for secretagogin was completely absent in 10 out of 16 homozygous tau tg mice (score 0, Fig. 1c), while in 4 cases sparse (score 1) and in 2 cases moderate (score 2) immunoreactivity was observed, respectively. The secretagogin positive cases showed no (n = 3) or only mild to moderate (n = 3) tau pathology, while moderate to severe tau pathology was detected in 8 out of 10 homozygous tau tg mice that lacked detectable hippocampal secretagogin immunoreactivity (Table 1). Secretagogin scores were significantly higher in wt mice compared to tau tg mice (Kruskal–Wallis H, P \ 0.001), while no significant difference was detected between hemizygous and homozygous tau tg mice (Fig. 2). Spearmans rank correlation revealed a significant negative correlation between secretagogin and tau scores (rho -0.619, P \ 0.01; Table 1). No influence of age or gender on the amount of secretagogin and tau immunoreactive neurones was detected (P [ 0.05). Table 1 Comparison of secretagogin and tau scores in tau transgenic (P301L) mice Secretagogin score
Fig. 2 Mean secretagogin immunoreactivity scores. For evaluation of secretagogin immunoreactivity in the hippocampus of 15 wt, 8 hemizygous and 16 homozygous tau tg mice we used a four-tired semiquantitative scoring system: no neuropil immunoreactivity and no positive neurones (score 0), some sparse neuropil immunoreactivity and/or some single positive neurones (score 1), moderate neuropil immunoreactivity and up to 50 positive neurones (score 2), widespread and strong neuropil immunoreactivity and over 50 positive neurones in the hippocampus (score 3). *P \ 0.001
tau score hemizygous tau tg (n = 8) 0
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2
homozygous tau tg (n = 16) 3
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0
1
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1
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0
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0
0
0
0
0
62.5
25
0
12.5
25
12.5
3 Total (%)
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0
43.75
18.75
Fig. 3 Absence of secretagogin and tau co-localisation. Double-immunofluorescence of secretagogin (a) and transgenic tau (b) in the granule cell layer of the dentate gyrus of P301L tau tg mice. Representative images and the overlay (c) are shown. Scalebar 100 lm
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We next determined co-localization of secretagogin and tau by double-immunofluorescence staining in hippocampus of three homozygous tau tg mice (Fig. 3). With the exception of only a very few neurones, staining for tau and secretagogin was mutually exclusive. We further examined secretagogin protein expression in wt and homozygous tau tg mice by Western blot analysis. In agreement with our immunohistochemical analyses, secretagogin expression was found to be reduced by 20–25% (P \ 0.01) in brains of homozygous tau tg mice when compared to age matched wt mice (Fig. 4). Finally, in a subset of cases (3 wt and 3 homozygous tau tg mice) we investigated secretagogin immunoreactivity in brain areas other than the hippocampus. We found secretagogin immunoreactivity in both genotyopes in
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glomerular, outer plexiform, mitral and inner plexiform layers and in outer parts of the granule cell layer of the olfactory bulb, respectively, as well as in bed nuclei of stria terminalis, lateral hypothalamic area of hypothalamus and superior colliculus (Fig. 5). Of note, in contrast to the hippocampus, those areas showed no differences regarding secretagogin immunoreactivity between wt and tau tg mice, partly reflecting the absence of P301L transgene expression in the olfactory bulb of P301L transgenic mice (Gotz et al. 2001, Fig. 5).
Discussion
Fig. 4 Western blots of secretagogin in hippocampal extracts. Analysis of 3 wt and 3 homozygous tau tg mice (P301L) demonstrating secretagogin expression to be significantly lower in homozygous tau tg compared to wt mice. a Representative immunoblot for secretagogin. GAPDH serves as loading control. b Quantification of immunoblotting for secretagogin. *P \ 0.05
In the present study, we demonstrate a role for tau in hippocampal secretagogin expression using tau tg mice. This clarifies previous findings on the relation between secretagogin expression and tau pathology in human AD. We have shown previously that hippocampal secretagogin immunoreactivity in humans with sporadic AD did not differ from secretagogin immunoreactivity observed in healthy controls (Attems et al. 2008). One could therefore assume that, in analogy to findings in humans, no differences in hippocampal secretagogin expression would be observed between wt and tau tg mice. However, in the present study we demonstrate that in both hemizygous and homozygous tau tg mice hippocampal secretagogin immunoreactivity is significantly lower than in wt mice, with 62.5% of homozygous tau tg mice completely lacking
Fig. 5 Secretagogin in parasagittal brain sections of wt and tau tg mice. Except for the hippocampal formation there are no differences in secretagogin immunoreactivity between wt (a–d) and homozygous tau tg mice (e–h). In parasagittal sections secretagogin immunoreactivity is seen in glomerular external plexiform, mitral, internal plexiform and granular layer of the olfactory bulb of wt (a) and homozygous tau tg mice (insert in e), in line with the absence of transgene expression in the olfactory bulb of P301L tau tg mice (Gotz et al. 2001). Likewise, bed nuclei of stria terminalis (b and f) lateral hypothalamic area of hypothalamus (c, c1, g and g1; c1, frame in c; g1, frame in g) and superior colliculus (d, d1, h and h1; d1, frame
in d; h1, frame in h) show secretagogin immunoreactivity in both wt (b–d) and homozygous tau tg mice (f–h). Hippocampal secretagogin immunoreactivity is seen in wt mice only (d) while homozygous tau tg mice lack detectable secretagogin immonoreactivity in the hippocampus (h). All sections were stained with a polyclonal secretagogin antibody (for details see ‘‘Immunohistiochemistry’’). OB olfactory bulb, gl glomerular layer, epl external plexiform layer, mi mitral layer, ipl internal plexiform layer, gr granule layer BST bed nuclei of stria terminalis, HY hypothalamus, LHA lateral hypothalamic area, HPF hippocampal formation, SC superior colliculus. Scalebars, 500 lm
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any detectable hippocampal secretagogin immunoreactivity. Moreover, by Western blot analysis we find that secretagogin levels are 20–25% reduced in the hippocampus of homozygous tau tg mice compared to wt. On the other hand, similar to data from human AD cases, where virtually no co-localizations between secretagogin and tau were seen in the hippocampus (Attems et al. 2008), double immunofluorescence in the present study revealed that secretagogin and tau expression in tau tg mice are mutually exclusive. As the P301L tau tg mice express the transgene in the hippocampus and not the olfactory bulb (Gotz et al. 2001), the latter serves as an appropriate internal control, showing unaltered secretagogin expression in tg olfactory bulb compared to wt. Overall, our data obtained in human AD cases and wt and tau tg mice strongly suggest an interdependency of secretagogin and tau. This raises the possibility that in sporadic, non-familial AD secretagogin expressing neurones are resistant to tau-associated neurodegeneration as suggested recently (Attems et al. 2008) while expression of tg tau above a certain threshold (e.g., P301L homozygous tau tg mice) impairs or down-regulates secretagogin expression in the hippocampus. Alternatively, this may reflect selective vulnerability and differences in expression in neuronal subpopulations in the hippocampus. In this study, we did not investigate AT100 phosphorylation that has been described in P301L tau tg mice (Gotz et al. 2001). It has been shown previously that AT100 is only phosphorylated in a small subset of neurones that are phosphorylated at the AT8 phospho-epitope (Deters et al. 2008) indicating that the number of AT100-positive neurones is much lower than the number of secretagogin-negative neurones (e.g., the entire hippocampal neuronal population in 62.5% of homozygous tau tg mice) in P301L tau tg mice, respectively. This indicates that tau accumulation but not phosphorylation of the pathological AT100 epitope negatively regulates secretagogin immunoreactivity. Our finding of secretagogin expression in the granule cell layer of the dentate gyrus in wt mice and in glomerular, outer plexiform, mitral and inner plexiform layers of the olfactory bulb in wt and tg mice is in line with data on secretagogin expression in mice and lemurs reported recently (Mulder et al. 2009). Mulder et al. found that secretagogin is a marker of neuroblasts commuting in the rostral migratory stream. The authors showed that secretagogin identifies granule cells in the dentate gyrus and terminally differentiated neurones in the olfactory bulb, where secretagogin is present in periglomerular cells and granular layer interneurones (Mulder et al. 2009). The cellular and molecular mechanisms behind the presumed neuroprotective effect(s) of secretagogin is (are) unknown. The lack of tau pathology in secretagogin expressing hippocampal neurones in human AD cases
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raised the possibility that secretagogin exerts neuroprotection (Attems et al. 2008), but those findings could not exclude the possibility that the lack of tau pathology in secretagogin expressing neurones merely represents an epiphenomenon rather than a causal relationship. However, together with data from recent in vitro studies (Maj et al. 2010), that suggested an interaction between secretagogin and tau, the significant reduction of hippocampal secretagogin expression in tau tg mice and the mutual exclusiveness of secretagogin and tau expression at the cellular level further strengthen the assumption of an interdependency of secretagogin and tau. Thereby, our data suggest that the lack of tau pathology in secretagogin expressing neurones in human AD cases probably is of functional relevance. Both in vitro and in vivo studies are required to determine whether secretagogin exerts a neuroprotective function in humans or whether tau negatively regulates secretagogin. Therefore, secretagogin could be a potential target for molecular based therapies of tau-associated neurodegeneration in AD. Acknowledgments This work was supported by the Newcastle NIHR Biomedical Research Centre In Ageing and Age Related Diseases, the Alzheimer Research Trust (ART-EG2010A-1) and by the Medical Scientific Fund of the Mayor of the City of Vienna (Grant number 08052). Mathias Heikenwalder was supported by the Prof. Dr. Max-Cloe¨tta foundation, the Hofschneider Stiftung and the Helmholtz-Zentrum Mu¨nchen. The authors thank Ms. Barbara Weidinger for excellent laboratory work.
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