- Email: [email protected]
Biochemical characterization of FTLD-TDP overexpression and knockout mouse models
P154
Oral O4-03: Animal and Cellular Models 2
of conformationally abnormal and hyperphosphorylated tau. The presence of NFT in the neocortex is associated with impairments of cognitive function, supporting a central role for tau in dementia. The significance of the accumulation of NFT for neuronal and cognitive function is still obscure. Objective (s): To investigate whether tau accumulation and aggregation is involved in inducing cognitive impairments by affecting synaptic function, we studied synaptic and cognitive functions of the htau mouse. The hTau mouse develops tau pathology similar to observed in AD in its distribution and its age-dependent development. Methods: Animals: htau mice and agematched wild type mice: male (C57BL/6 J) ages 4 and 12 months. Electrophysiology: extracellular field excitatory postsynaptic potentials (EPSPs) evoked at the CA3-CA1 synapse were recorded to assess basal synaptic transmission (input-output curve and paired pulse facilitation) and long term potentiation (LTP). Behavior: visual recognition memory was measured by an object recognition memory test that measured spontaneous preference for a novel object compared to a familiar object. The spatial memory was assessed by the Morris water maze test (MWM). Results: Htau mice develop age-dependent cognitive and physiological impairments. 4 months-old htau mice, with early stage of tau pathology (pre-tangles) are unaffected behaviorally and physiologically. Concurrent with the development of moderate tau pathology in the forebrain, 12 months-old htau mice develop learning and memory deficits. 12 months-old htau mice show cognitive deficits in object recognition and spatial memories which are associated with cortical and hippocampal dysfunction, precisely the brain regions affected by tau pathology. Basal synaptic transmission and induction of LTP with high frequency stimulation (HFS), but not theta burst stimulation, are perturbed in hippocampal CA1 region of old but not young htau mice. In addition, at 12 months htau mice had diminished charge transfer during HFS as a result of the inability of Schaffer collateral fibers to sustain high frequency activity. Conclusions: Our results suggest that tau pathology may underlie an age-dependent learning impairment through disruption of synaptic function. O4-03-02
ACTIVATED ASTROCYTES SUPPRESS AMYLOID PLAQUE PATHOGENESIS IN APP/PS1 MICE
Andrew W. Kraft1, Xiaoyan Hu1, Ping Yan1, Qingli Xiao1, Brandon B. Holmes1, Ulrika Wilhelmsson2, John R. Cirrito1, David M. Holtzman1, Milos Pekny2, Jin-Moo Lee1, 1Washington University in St. Louis, St. Louis, MO, USA; 2Go¨teborg University, Go¨teborg, Sweden. Contact e-mail: [email protected] Background: Amyloid plaques and associated reactive gliosis are hallmark pathological features of Alzheimer’s disease (AD). Activated microglia are believed to be play a role in the direct phagocytosis (albeit impaired) of amyloid, suppressing plaque progression; however, the precise role of activated astrocytes is not well-defined. Methods: To examine the role of astrocytosis in AD, we deleted the genes of two intermediate filaments required for astrocyte activation-glial fibrillary acid protein (GFAP) and vimentin (Vim)-in APP/PS1 mice. Previous studies have shown that gene deletion of gfap and vim results in normal-appearing astrocytes that are incapable of producing a reactive phenotype in response to brain injury or inflammation. Plaque load (% area), number, and size (um2) were compared between APP/PS1 gfap-/- vim-/- (g-v-) and APP/PS1 gfapþ/þ vimþ/þ (gþvþ) mice, using a high-resolution ‘‘virtual microscopy’’ digital scanner and Image J software (to delineate plaques). Ab levels in brain tissue was determined using ELISA. Astrocyte morphology in these two genotypes was examined by injecting mice with lentiviral vectors carrying green fluorescent protein (GFP) driven by a GFAP promoter 2 weeks prior to microscopic examination. Results: APP/PS1 g-v- mice had significantly greater plaque burden than APP/PS1 gþvþ mice at 8 and 12 months of age, but not at 4 months of age (very early in plaque pathogenesis). Cortical plaque load was 0.19% in g-v- mice vs. 0.22% in gþvþ mice at 4 months (n.s.), 4.15% vs. 1.96% at 8 months (p < 0.05), and 9.73% vs. 5.63% (p < 0.05). Corroborating the plaque load data, guanidine extracts from g-v- brains showed greater Abx-42 levels than in gþvþ brains (527.90 ng Ab /mg protein vs 755.74 ng Ab /mg protein, p < 0.05), while PBS and triton extracts were similar. Furthermore, the distribution of plaque sizes was altered by gfap/vim gene deletion: g-v- mice had more plaques of larger size. Astrocytes in both gþvþ and g-v- mice were
closely associated with plaques; however, g-v- astrocytes showed finer processes and demonstrated attenuation of hypertrophic processes compared to gþvþ astrocytes. Conclusions: These results suggest that astrocyte activation does not affect initial plaque deposition, but limits subsequent progression of amyloid plaque pathogenesis. O4-03-03
BIOCHEMICAL CHARACTERIZATION OF FTLD-TDP OVEREXPRESSION AND KNOCKOUT MOUSE MODELS
Hans Wils1, Gernot Kleinberger2, Geert Joris2, Ivy Cuijt2, Christine Van Broeckhoven2, Samir Kumar-Singh2, 1Neurodegenerative Brain Diseases Group, VIB - Department of Molecular Genetics, University of Antwerp, Antwerpen, Belgium; 2Neurodegenerative Brain Diseases Group, Department of Molecular Genetics, VIB, University of Antwerp, Antwerpen, Belgium. Contact e-mail: [email protected] Background: Overexpression of progranulin (GRN) is involved in tumorigenesis, while reduced levels of GRN lead to frontotemporal dementia with ubiquitin-positive neuronal cytoplasmic and intranuclear inclusions. Phosphorylated forms of the TAR DNA binding protein (TDP-43) were identified as the major protein constituent of these inclusions (FTLD-TDP). However, the mechanisms underlying GRN-mediated cell proliferation and neuronal survival are still unclear. Recent studies have suggested that siRNA mediated knockdown of GRN leads to TDP-43 fragmentation and apoptosis due to caspase-3 activation. Methods: To gain further insights in the mechanism of FTLD-TDP and the interaction between GRN and TDP-43, we developed a series of transgenic overexpression and conditional knockout mice. For instance, to study GRN haploinsufficiency-led neurodegeneration, a conditional Grn knockout mouse was generated. Also, mice expressing wildtype human GRN, or TDP-43 wildtype or ALS-mutant (M337 V) under the transcriptional control of a modified mThy-1 promoter were generated. Results: Western blot analysis on 6 months old Grn knockout mice showed no difference in TDP-43 fragmentation pattern or caspase-3 activation in homozygous knockout mice compared to nontransgenic littermate controls. Also, on immunohistochemistry, we did not observe apparent differences for TDP-43 and caspase-3 staining between these mice. We are also analyzing and comparing these data with wildtype GRN overexpression mice where we generated several transgenic lines with at least 2 mice lines having more than 4 times overexpression compared to the endogenous protein. Interestingly, both TDP-43 wild type and mutant mice lines had much lower protein expression compared to endogenous TDP-43. The maximal transgenic expression for wildtype TDP-43 was z0.5x while for mutant TDP-43 this was slightly lower than 1x endogenous TDP-43 expression level. Conclusions: While studies are ongoing, data from Grn knockout mice already suggest that although caspase-3 activation can lead to TDP-43 cleavage and aggregation in vitro, it is most likely not the cause of TDP-43 fragmentation in vivo. Also, consistently lower expression levels achieved for TDP-43 transgenic mice suggest that TDP-43 expression is tightly controlled. These mice lines will be very important for understanding the physiological function and the potential link between GRN haploinsufficiency and TDP-43 pathology. O4-03-04
POSSIBLE MUTUAL INTERACTION BETWEEN DIABETES MELLITUS AND ALZHEIMER’S DISEASE: GENERATION OF NOVEL TRANSGENIC MICE MODELS OF ALZHEIMER’S DISEASE WITH DIABETIC CONDITIONS
Shuko Takeda1, Naoyuki Sato1, Kozue Uchio2, Kyoko Sawada2, Takanori Kunieda2, Hiromi Rakugi1, Ryuichi Morishita1, 1Osaka University, Osaka, Japan; 2Laboratory Animal Resource Bank, National Institute of Biomedical Innovation, osaka, Japan. Contact e-mail: [email protected]. osaka-u.ac.jp Background: Recent epidemiological studies suggest that diabetes mellitus are among the risk factors for Alzheimer’s disease (AD). However, the molecular bases that link diabetes with AD are unclear. In this study, to examine the pathophysiological interaction between diabetes mellitus and AD in vivo, we generated two types of transgenic mice which show pathological
Oral O4-03: Animal and Cellular Models 2
of conformationally abnormal and hyperphosphorylated tau. The presence of NFT in the neocortex is associated with impairments of cognitive function, supporting a central role for tau in dementia. The significance of the accumulation of NFT for neuronal and cognitive function is still obscure. Objective (s): To investigate whether tau accumulation and aggregation is involved in inducing cognitive impairments by affecting synaptic function, we studied synaptic and cognitive functions of the htau mouse. The hTau mouse develops tau pathology similar to observed in AD in its distribution and its age-dependent development. Methods: Animals: htau mice and agematched wild type mice: male (C57BL/6 J) ages 4 and 12 months. Electrophysiology: extracellular field excitatory postsynaptic potentials (EPSPs) evoked at the CA3-CA1 synapse were recorded to assess basal synaptic transmission (input-output curve and paired pulse facilitation) and long term potentiation (LTP). Behavior: visual recognition memory was measured by an object recognition memory test that measured spontaneous preference for a novel object compared to a familiar object. The spatial memory was assessed by the Morris water maze test (MWM). Results: Htau mice develop age-dependent cognitive and physiological impairments. 4 months-old htau mice, with early stage of tau pathology (pre-tangles) are unaffected behaviorally and physiologically. Concurrent with the development of moderate tau pathology in the forebrain, 12 months-old htau mice develop learning and memory deficits. 12 months-old htau mice show cognitive deficits in object recognition and spatial memories which are associated with cortical and hippocampal dysfunction, precisely the brain regions affected by tau pathology. Basal synaptic transmission and induction of LTP with high frequency stimulation (HFS), but not theta burst stimulation, are perturbed in hippocampal CA1 region of old but not young htau mice. In addition, at 12 months htau mice had diminished charge transfer during HFS as a result of the inability of Schaffer collateral fibers to sustain high frequency activity. Conclusions: Our results suggest that tau pathology may underlie an age-dependent learning impairment through disruption of synaptic function. O4-03-02
ACTIVATED ASTROCYTES SUPPRESS AMYLOID PLAQUE PATHOGENESIS IN APP/PS1 MICE
Andrew W. Kraft1, Xiaoyan Hu1, Ping Yan1, Qingli Xiao1, Brandon B. Holmes1, Ulrika Wilhelmsson2, John R. Cirrito1, David M. Holtzman1, Milos Pekny2, Jin-Moo Lee1, 1Washington University in St. Louis, St. Louis, MO, USA; 2Go¨teborg University, Go¨teborg, Sweden. Contact e-mail: [email protected] Background: Amyloid plaques and associated reactive gliosis are hallmark pathological features of Alzheimer’s disease (AD). Activated microglia are believed to be play a role in the direct phagocytosis (albeit impaired) of amyloid, suppressing plaque progression; however, the precise role of activated astrocytes is not well-defined. Methods: To examine the role of astrocytosis in AD, we deleted the genes of two intermediate filaments required for astrocyte activation-glial fibrillary acid protein (GFAP) and vimentin (Vim)-in APP/PS1 mice. Previous studies have shown that gene deletion of gfap and vim results in normal-appearing astrocytes that are incapable of producing a reactive phenotype in response to brain injury or inflammation. Plaque load (% area), number, and size (um2) were compared between APP/PS1 gfap-/- vim-/- (g-v-) and APP/PS1 gfapþ/þ vimþ/þ (gþvþ) mice, using a high-resolution ‘‘virtual microscopy’’ digital scanner and Image J software (to delineate plaques). Ab levels in brain tissue was determined using ELISA. Astrocyte morphology in these two genotypes was examined by injecting mice with lentiviral vectors carrying green fluorescent protein (GFP) driven by a GFAP promoter 2 weeks prior to microscopic examination. Results: APP/PS1 g-v- mice had significantly greater plaque burden than APP/PS1 gþvþ mice at 8 and 12 months of age, but not at 4 months of age (very early in plaque pathogenesis). Cortical plaque load was 0.19% in g-v- mice vs. 0.22% in gþvþ mice at 4 months (n.s.), 4.15% vs. 1.96% at 8 months (p < 0.05), and 9.73% vs. 5.63% (p < 0.05). Corroborating the plaque load data, guanidine extracts from g-v- brains showed greater Abx-42 levels than in gþvþ brains (527.90 ng Ab /mg protein vs 755.74 ng Ab /mg protein, p < 0.05), while PBS and triton extracts were similar. Furthermore, the distribution of plaque sizes was altered by gfap/vim gene deletion: g-v- mice had more plaques of larger size. Astrocytes in both gþvþ and g-v- mice were
closely associated with plaques; however, g-v- astrocytes showed finer processes and demonstrated attenuation of hypertrophic processes compared to gþvþ astrocytes. Conclusions: These results suggest that astrocyte activation does not affect initial plaque deposition, but limits subsequent progression of amyloid plaque pathogenesis. O4-03-03
BIOCHEMICAL CHARACTERIZATION OF FTLD-TDP OVEREXPRESSION AND KNOCKOUT MOUSE MODELS
Hans Wils1, Gernot Kleinberger2, Geert Joris2, Ivy Cuijt2, Christine Van Broeckhoven2, Samir Kumar-Singh2, 1Neurodegenerative Brain Diseases Group, VIB - Department of Molecular Genetics, University of Antwerp, Antwerpen, Belgium; 2Neurodegenerative Brain Diseases Group, Department of Molecular Genetics, VIB, University of Antwerp, Antwerpen, Belgium. Contact e-mail: [email protected] Background: Overexpression of progranulin (GRN) is involved in tumorigenesis, while reduced levels of GRN lead to frontotemporal dementia with ubiquitin-positive neuronal cytoplasmic and intranuclear inclusions. Phosphorylated forms of the TAR DNA binding protein (TDP-43) were identified as the major protein constituent of these inclusions (FTLD-TDP). However, the mechanisms underlying GRN-mediated cell proliferation and neuronal survival are still unclear. Recent studies have suggested that siRNA mediated knockdown of GRN leads to TDP-43 fragmentation and apoptosis due to caspase-3 activation. Methods: To gain further insights in the mechanism of FTLD-TDP and the interaction between GRN and TDP-43, we developed a series of transgenic overexpression and conditional knockout mice. For instance, to study GRN haploinsufficiency-led neurodegeneration, a conditional Grn knockout mouse was generated. Also, mice expressing wildtype human GRN, or TDP-43 wildtype or ALS-mutant (M337 V) under the transcriptional control of a modified mThy-1 promoter were generated. Results: Western blot analysis on 6 months old Grn knockout mice showed no difference in TDP-43 fragmentation pattern or caspase-3 activation in homozygous knockout mice compared to nontransgenic littermate controls. Also, on immunohistochemistry, we did not observe apparent differences for TDP-43 and caspase-3 staining between these mice. We are also analyzing and comparing these data with wildtype GRN overexpression mice where we generated several transgenic lines with at least 2 mice lines having more than 4 times overexpression compared to the endogenous protein. Interestingly, both TDP-43 wild type and mutant mice lines had much lower protein expression compared to endogenous TDP-43. The maximal transgenic expression for wildtype TDP-43 was z0.5x while for mutant TDP-43 this was slightly lower than 1x endogenous TDP-43 expression level. Conclusions: While studies are ongoing, data from Grn knockout mice already suggest that although caspase-3 activation can lead to TDP-43 cleavage and aggregation in vitro, it is most likely not the cause of TDP-43 fragmentation in vivo. Also, consistently lower expression levels achieved for TDP-43 transgenic mice suggest that TDP-43 expression is tightly controlled. These mice lines will be very important for understanding the physiological function and the potential link between GRN haploinsufficiency and TDP-43 pathology. O4-03-04
POSSIBLE MUTUAL INTERACTION BETWEEN DIABETES MELLITUS AND ALZHEIMER’S DISEASE: GENERATION OF NOVEL TRANSGENIC MICE MODELS OF ALZHEIMER’S DISEASE WITH DIABETIC CONDITIONS
Shuko Takeda1, Naoyuki Sato1, Kozue Uchio2, Kyoko Sawada2, Takanori Kunieda2, Hiromi Rakugi1, Ryuichi Morishita1, 1Osaka University, Osaka, Japan; 2Laboratory Animal Resource Bank, National Institute of Biomedical Innovation, osaka, Japan. Contact e-mail: [email protected]. osaka-u.ac.jp Background: Recent epidemiological studies suggest that diabetes mellitus are among the risk factors for Alzheimer’s disease (AD). However, the molecular bases that link diabetes with AD are unclear. In this study, to examine the pathophysiological interaction between diabetes mellitus and AD in vivo, we generated two types of transgenic mice which show pathological