- Email: [email protected]
Trehalose enhances the degradation of Tau through the autophagy pathway
S556 P3-129
Poster Presentations P3 TIME-LINE OF PATHOLOGY AND DEFECTS IN BIAT MICE WITH COMBINED AMYLOID-TAU PATHOLOGY 1
1
1
1
Anna Kremer , Herve Maurin , Benoit Lechat , Peter Borghgraef , Herman Devijver1, Fred Van Leuven2, 1University of Leuven, Leuven, Belgium; 2 Experimental Genetics group - LEGTEGG - KULeuven, Leuven, Belgium. Background: Alzheimer’s disease (AD) post-mortem pathology consists of amyloid plaques, neurofibrillary tangles and neuropil treads, but their relation to early clinical and functional defects remains not precisely defined in humans. In mouse models, the effects of APP and Tau expression in and on neurons can be studied in spatial and temporal resolution, unattainable in human brain. Thereby the mouse models render important insights into AD, notwithstanding the necessary and evident caveats inherent to modelbuilding. The relation between the different pathologies and the functional defects are assessed, and eventually therapeutically addressed by us in mouse models that present either amyloid or tau pathology, or both combined, all in the same genetic background. Methods: We defined the comprehensive time-line of cognitive defects and pathological traits of bigenic APP.V717IxTau.P301L (biAT) mice, relative to their parental single transgenic strains suffering amyloid or tau pathology separately. In addition, biAT mice are comparatively analyzed with bigenic GSK3ßxTau.P301L mice (biGT) expressing the identical Tau.P301L transgene in combination with the GSK3ß[S9A] transgene (Spittaels et al, 2000; Terwel et al, 2008). All genotypes are in the same FvB/N genetic background. While the late pathology in the bigenic models is now well analyzed and understood (Muyllaert et al, 2006; Terwel et al, 2008) the early defects and their inter-relations remain open for experimental analysis. Results: The bigenic biAT mice present with increased phosphorylation of protein tau already at young age (2-4 months) when neurons became burdened with intracellular amyloid (icAß). Both beginning pathologies co-exist in the same neurons in cortex and hippocampus. Remarkably, dendritic spine density is increased at 4-6 months while LTP and cognition are impaired in biAT mice, similar to the parental APP.V717I mice (Moechars et al, 1999). At this age, in both APP.V717I and biAT mice the GSK3 isozymes are activated as indicated by increased tyrosine auto-phosphorylation (pY279/pY216-GSK3a/b). The activation of the GSK3 kinases coincides with the augmented phosphorylation of protein Tau, resulting in aggravated tauopathy in cortex and hippocampus of biAT mice compared to Tau.P301L mice. GSK3 is thereby confirmed as major link from amyloid to tau pathology (Muyllaert et al, 2007; Terwel et al, 2008). Observations on large cohorts (65 M/74 F) in our expanded colony of biAT mice, revealed important mortality in the age-window of 3-6 months in males and females alike, with less than 50% of biAT mice surviving over 6 months. In general, female biAT mice survive markedly longer (some up to 20-22 months) than males that do not become older than 13-14 months. This is in sharp contrast with the parental Tau.P301L mice that die without exception before age 12 months (mean 9.4 months, no gender difference). The analogy with the prolonged survival of the biGT mice is thereby corroborated (Terwel et al, 2008), underlining the hypothesis that GSK3 increases phosphorylation of protein tau and promotes its aggregation. This then is concluded to be beneficial at least for the respiratory control centers in the brain-stem (Dutschmann et al, 2010), an aspect of interest that we are analyzing in depth. A remarkable point is the early and progressive motor problems of the biGT mice that are much more severe than in the biAT mice or parental Tau.P301L mice. They necessitates feeding of biGT mice on the cage-floor from early age onwards, which nevertheless allows them to survive longer than Tau.P301L and biAT mice, making the case of GSK3 as controlling protein tau phosphorylation even stronger. Conclusions: Our continued studies of young and old, single and bigenic amyloid and tau transgenic mice define important analogies and major differences in functional, biochemical and pathological aspects of their AD-related phenotypes. The activation of GSK3 kinases by amyloid and the resulting phosphorylation of protein Tau is a most early event in the biAT model. The elucidation of the mechanism by which Ab increases tyrosine auto-phosphorylation of GSK3a/b is a fundamental biochemical and cell-biological challenge with far-reaching physiological and pathological implications.
P3-130
TREHALOSE ENHANCES THE DEGRADATION OF TAU THROUGH THE AUTOPHAGY PATHWAY
Ulrike Krueger*1, Yipeng Wang1, Xiaoyu Li1, Eva Mandelkow1, 1 Max-Planck Institute, Hamburg, Germany. Background: Modulating the Tau level may represent a therapeutic target for Alzheimer Disease, as accumulating evidence shows that Abeta-induced neurodegeneration is mediated by Tau. It is therefore important to understand the expression and degradation of Tau in neurons. Recently we showed that over expressed mutant Tau and Tau aggregates are degraded via the autophagic pathway in an N2a cell model, and how autophagy contributes to Tau cleavage, aggregation, and degradation (Wang et al., HMG2009). Here we investigated how different aspects of autophagy contribute to the degradation of Tau in cultured primary neurons. We further examined whether the stimulation of autophagy can reduce aggregation and alleviate cytotoxicity in a regulable N2a cell model of Tauopathy. Methods: Autophagy was induced by Trehalose in Neuroblastoma cells and rat primary neurons. The activation of autophagy was validated by Flux assays and fluorescence microscopy. Tau level was analysed by western blot with a pan-Tau antibody and a series of phosphorylation dependent antibodies: 12E8, PHF1, AT8 and AT180.Tau aggregates were separated with Sarkosyl extraction and visualized with Thioflavin S staining. Results: Trehalose activated autophagy in rat cortical neurons, as seen by (1) elevated LC3-II levels, (2) an increased number of autophagosomes, (3) stimulated flux and (4) reduced p62 level, a known substrate of autophagy. At the same time Trehalose significantly reduced the endogenous Tau level. Phosphorylation seems to have no effect on Tau degradation by autophagy, as Tau phosphorylated at 12E8, PHF1, AT8 and AT180 sites was also decreased upon Trehalose treatment. In a N2a cell model of Tauopathy, Trehalose induced autophagy, which caused the reduction of Tau aggregation and toxicity. Conclusions: The activation of autophagy reduced endogenous Tau regardless of phosphorylation in primary cortical neurons and Tau aggregates in a N2a cell model of Tauopathy, indicating that stimulation of autophagy could serve as a therapeutic target. P3-131
RELATIONSHIP OF TUMOR-PROMOTING FACTORS LAPTM4B AND ALZHEIMER’S DISEASE PATHOLOGIES
Chuitxia Li1, Zhi Tang1, Bengt Winblad1, Rouli Zhou2, Jin Jing1, 1Karolinska instituted Stockholm, Sweden; 2Peking University, Beijing, China. Background: The mechanism how neurofibrillary tangles (NFTs) and senile plaques (SPs) are formed in Alzheimer’s disease (AD) brain is not clear. The human lysosomal protein transmembrane 4 beta is a novel hepatocellular carcinoma-associated gene. LAPTM4B is a type III transmembrane protein with four putative transmembrane regions. The isoform LAPTM4B- 24 misses 91 amino acids in the N-terminal than the isoform LAPTM4B-35, the longer isoform inhibits cell apoptosis, promotes cell proliferation, migration and invasion, while the shorter isoform inhibits cell survival or growth. LAPTM4B is mainly localized on plasma membrane and membranous organelles including endosomes and lysosomes, which has recently been proposed as candidate subcellular sites for AD pathogenesis. Meanwhile, LAPTM4B-35 activates PI3K-AKT signaling pathway through interaction of proline-rich motif in the N- terminus of LAPTM4B-35 with the p85a regulatory subunit of PI3K. PI3K-AKT signaling pathway plays an important role in AD pathologies Methods: The polyclonal antibody (pAb) Nl- 99 LAPTM4B that specifically recognizes LAPTM4B-35, pAb EC2 to LAPTM4B that recognizes both LAPTM4B-35 and LAPTM4B-24 isoforms were used to immunostain paraffin- embedded sections of post-mortem human brains. Results: We observed a dramatically reduced intensity and number of neurons positive for both Nl-99 and EC2 in the CA1 and CA4 of the hippocampus, as well as in the pyramidal neurons of the layers III and V in the temporal cortex in 6 AD as compared to 6 control cases. Using antibodies to phospho-tau (PHF-1 and pAb S422) as markers to NFTs, our double immunostaining data revealed that most normal looking neurons show a stronger immunoreactivity of LAPTM4B than tangle bearing neurons labeled by PHF-1 or pS422. The immunostainings of Nl-99 and EC2 are relatively weaker in most tangle bearing neurons including those with a few
Poster Presentations P3 TIME-LINE OF PATHOLOGY AND DEFECTS IN BIAT MICE WITH COMBINED AMYLOID-TAU PATHOLOGY 1
1
1
1
Anna Kremer , Herve Maurin , Benoit Lechat , Peter Borghgraef , Herman Devijver1, Fred Van Leuven2, 1University of Leuven, Leuven, Belgium; 2 Experimental Genetics group - LEGTEGG - KULeuven, Leuven, Belgium. Background: Alzheimer’s disease (AD) post-mortem pathology consists of amyloid plaques, neurofibrillary tangles and neuropil treads, but their relation to early clinical and functional defects remains not precisely defined in humans. In mouse models, the effects of APP and Tau expression in and on neurons can be studied in spatial and temporal resolution, unattainable in human brain. Thereby the mouse models render important insights into AD, notwithstanding the necessary and evident caveats inherent to modelbuilding. The relation between the different pathologies and the functional defects are assessed, and eventually therapeutically addressed by us in mouse models that present either amyloid or tau pathology, or both combined, all in the same genetic background. Methods: We defined the comprehensive time-line of cognitive defects and pathological traits of bigenic APP.V717IxTau.P301L (biAT) mice, relative to their parental single transgenic strains suffering amyloid or tau pathology separately. In addition, biAT mice are comparatively analyzed with bigenic GSK3ßxTau.P301L mice (biGT) expressing the identical Tau.P301L transgene in combination with the GSK3ß[S9A] transgene (Spittaels et al, 2000; Terwel et al, 2008). All genotypes are in the same FvB/N genetic background. While the late pathology in the bigenic models is now well analyzed and understood (Muyllaert et al, 2006; Terwel et al, 2008) the early defects and their inter-relations remain open for experimental analysis. Results: The bigenic biAT mice present with increased phosphorylation of protein tau already at young age (2-4 months) when neurons became burdened with intracellular amyloid (icAß). Both beginning pathologies co-exist in the same neurons in cortex and hippocampus. Remarkably, dendritic spine density is increased at 4-6 months while LTP and cognition are impaired in biAT mice, similar to the parental APP.V717I mice (Moechars et al, 1999). At this age, in both APP.V717I and biAT mice the GSK3 isozymes are activated as indicated by increased tyrosine auto-phosphorylation (pY279/pY216-GSK3a/b). The activation of the GSK3 kinases coincides with the augmented phosphorylation of protein Tau, resulting in aggravated tauopathy in cortex and hippocampus of biAT mice compared to Tau.P301L mice. GSK3 is thereby confirmed as major link from amyloid to tau pathology (Muyllaert et al, 2007; Terwel et al, 2008). Observations on large cohorts (65 M/74 F) in our expanded colony of biAT mice, revealed important mortality in the age-window of 3-6 months in males and females alike, with less than 50% of biAT mice surviving over 6 months. In general, female biAT mice survive markedly longer (some up to 20-22 months) than males that do not become older than 13-14 months. This is in sharp contrast with the parental Tau.P301L mice that die without exception before age 12 months (mean 9.4 months, no gender difference). The analogy with the prolonged survival of the biGT mice is thereby corroborated (Terwel et al, 2008), underlining the hypothesis that GSK3 increases phosphorylation of protein tau and promotes its aggregation. This then is concluded to be beneficial at least for the respiratory control centers in the brain-stem (Dutschmann et al, 2010), an aspect of interest that we are analyzing in depth. A remarkable point is the early and progressive motor problems of the biGT mice that are much more severe than in the biAT mice or parental Tau.P301L mice. They necessitates feeding of biGT mice on the cage-floor from early age onwards, which nevertheless allows them to survive longer than Tau.P301L and biAT mice, making the case of GSK3 as controlling protein tau phosphorylation even stronger. Conclusions: Our continued studies of young and old, single and bigenic amyloid and tau transgenic mice define important analogies and major differences in functional, biochemical and pathological aspects of their AD-related phenotypes. The activation of GSK3 kinases by amyloid and the resulting phosphorylation of protein Tau is a most early event in the biAT model. The elucidation of the mechanism by which Ab increases tyrosine auto-phosphorylation of GSK3a/b is a fundamental biochemical and cell-biological challenge with far-reaching physiological and pathological implications.
P3-130
TREHALOSE ENHANCES THE DEGRADATION OF TAU THROUGH THE AUTOPHAGY PATHWAY
Ulrike Krueger*1, Yipeng Wang1, Xiaoyu Li1, Eva Mandelkow1, 1 Max-Planck Institute, Hamburg, Germany. Background: Modulating the Tau level may represent a therapeutic target for Alzheimer Disease, as accumulating evidence shows that Abeta-induced neurodegeneration is mediated by Tau. It is therefore important to understand the expression and degradation of Tau in neurons. Recently we showed that over expressed mutant Tau and Tau aggregates are degraded via the autophagic pathway in an N2a cell model, and how autophagy contributes to Tau cleavage, aggregation, and degradation (Wang et al., HMG2009). Here we investigated how different aspects of autophagy contribute to the degradation of Tau in cultured primary neurons. We further examined whether the stimulation of autophagy can reduce aggregation and alleviate cytotoxicity in a regulable N2a cell model of Tauopathy. Methods: Autophagy was induced by Trehalose in Neuroblastoma cells and rat primary neurons. The activation of autophagy was validated by Flux assays and fluorescence microscopy. Tau level was analysed by western blot with a pan-Tau antibody and a series of phosphorylation dependent antibodies: 12E8, PHF1, AT8 and AT180.Tau aggregates were separated with Sarkosyl extraction and visualized with Thioflavin S staining. Results: Trehalose activated autophagy in rat cortical neurons, as seen by (1) elevated LC3-II levels, (2) an increased number of autophagosomes, (3) stimulated flux and (4) reduced p62 level, a known substrate of autophagy. At the same time Trehalose significantly reduced the endogenous Tau level. Phosphorylation seems to have no effect on Tau degradation by autophagy, as Tau phosphorylated at 12E8, PHF1, AT8 and AT180 sites was also decreased upon Trehalose treatment. In a N2a cell model of Tauopathy, Trehalose induced autophagy, which caused the reduction of Tau aggregation and toxicity. Conclusions: The activation of autophagy reduced endogenous Tau regardless of phosphorylation in primary cortical neurons and Tau aggregates in a N2a cell model of Tauopathy, indicating that stimulation of autophagy could serve as a therapeutic target. P3-131
RELATIONSHIP OF TUMOR-PROMOTING FACTORS LAPTM4B AND ALZHEIMER’S DISEASE PATHOLOGIES
Chuitxia Li1, Zhi Tang1, Bengt Winblad1, Rouli Zhou2, Jin Jing1, 1Karolinska instituted Stockholm, Sweden; 2Peking University, Beijing, China. Background: The mechanism how neurofibrillary tangles (NFTs) and senile plaques (SPs) are formed in Alzheimer’s disease (AD) brain is not clear. The human lysosomal protein transmembrane 4 beta is a novel hepatocellular carcinoma-associated gene. LAPTM4B is a type III transmembrane protein with four putative transmembrane regions. The isoform LAPTM4B- 24 misses 91 amino acids in the N-terminal than the isoform LAPTM4B-35, the longer isoform inhibits cell apoptosis, promotes cell proliferation, migration and invasion, while the shorter isoform inhibits cell survival or growth. LAPTM4B is mainly localized on plasma membrane and membranous organelles including endosomes and lysosomes, which has recently been proposed as candidate subcellular sites for AD pathogenesis. Meanwhile, LAPTM4B-35 activates PI3K-AKT signaling pathway through interaction of proline-rich motif in the N- terminus of LAPTM4B-35 with the p85a regulatory subunit of PI3K. PI3K-AKT signaling pathway plays an important role in AD pathologies Methods: The polyclonal antibody (pAb) Nl- 99 LAPTM4B that specifically recognizes LAPTM4B-35, pAb EC2 to LAPTM4B that recognizes both LAPTM4B-35 and LAPTM4B-24 isoforms were used to immunostain paraffin- embedded sections of post-mortem human brains. Results: We observed a dramatically reduced intensity and number of neurons positive for both Nl-99 and EC2 in the CA1 and CA4 of the hippocampus, as well as in the pyramidal neurons of the layers III and V in the temporal cortex in 6 AD as compared to 6 control cases. Using antibodies to phospho-tau (PHF-1 and pAb S422) as markers to NFTs, our double immunostaining data revealed that most normal looking neurons show a stronger immunoreactivity of LAPTM4B than tangle bearing neurons labeled by PHF-1 or pS422. The immunostainings of Nl-99 and EC2 are relatively weaker in most tangle bearing neurons including those with a few