Neurofibrillary and neurodegenerative pathology in APP-transgenic mice injected with AAV2-mutant TAU: neuroprotective effects of Cerebrolysin.
ABSTRACT: Alzheimer's disease (AD) continues to be the most common cause of cognitive and motor alterations in the aging population. Accumulation of amyloid beta (Abeta)-protein oligomers and the microtubule associated protein-TAU might be responsible for the neurological damage. We have previously shown that Cerebrolysin (CBL) reduces the synaptic and behavioral deficits in amyloid precursor protein (APP) transgenic (tg) mice by decreasing APP phosphorylation via modulation of glycogen synthase kinase-3beta (GSK3beta) and cyclin-dependent kinase-5 (CDK5) activity. These kinases also regulate TAU phosphorylation and are involved in promoting neurofibrillary pathology. In order to investigate the neuroprotective effects of CBL on TAU pathology, a new model for neurofibrillary alterations was developed using somatic gene transfer with adeno-associated virus (AAV2)-mutant (mut) TAU (P301L). The Thy1-APP tg mice (3 m/o) received bilateral injections of AAV2-mutTAU or AAV2-GFP, into the hippocampus. After 3 months, compared to non-tg controls, in APP tg mice intra-hippocampal injections with AAV2-mutTAU resulted in localized increased accumulation of phosphorylated TAU and neurodegeneration. Compared with vehicle controls, treatment with CBL in APP tg injected with AAV2-mutTAU resulted in a significant decrease in the levels of TAU phosphorylation at critical sites dependent on GSK3beta and CDK5 activity. This was accompanied by amelioration of the neurodegenerative alterations in the hippocampus. This study supports the concept that the neuroprotective effects of CBL may involve the reduction of TAU phosphorylation by regulating kinase activity.
Project description:The glycogen synthase kinase-3beta (GSK3beta) pathway plays an important role in mediating neuronal fate and synaptic plasticity. In Alzheimer's disease (AD), abnormal activation of this pathway might play an important role in neurodegeneration, and compounds such as lithium that modulate GSK3beta activity have been shown to reduce amyloid production and tau phosphorylation in amyloid precursor protein (APP) transgenic (tg) mice. However, it is unclear whether regulation of GSK3beta is neuroprotective in APP tg mice. In this context, the main objective of the present study was to determine whether pharmacological or genetic manipulations that block the GSK3beta pathway might ameliorate the neurodegenerative alterations in APP tg mice and to better understand the mechanisms involved. For this purpose, two sets of experiments were performed. First, tg mice expressing mutant human APP under the Thy1 promoter (hAPP tg) were treated with either lithium chloride or saline alone. Second, hAPP tg mice were crossed with GSK3beta tg mice, in which overexpression of this signaling molecule results in a dominant-negative (DN) effect with inhibition of activity. hAPP tg mice that were treated with lithium or that were crossed with DN-GSK3beta tg mice displayed improved performance in the water maze, preservation of the dendritic structure in the frontal cortex and hippocampus, and decreased tau phosphorylation. Moreover, reduced activation of GSK3beta was associated with decreased levels of APP phosphorylation that resulted in decreased amyloid-beta production. In conclusion, the present study showed that modulation of the GSK3beta signaling pathway might also have neuroprotective effects in tg mice by regulating APP maturation and processing and further supports the notion that GSK3beta might be a suitable target for the treatment of AD.
Project description:Alzheimer's disease (AD) is reportedly associated with the accumulation of calcium ions (Ca2+), and this accumulation is responsible for the phosphorylation of tau. Although several lines of evidence demonstrate the above phenomenon, the inherent mechanisms remain unknown. Using APP/PS1 Tg mice and neuroblastoma (N)2a cells as in vivo and in vitro experimental models, we observed that Ca2+ stimulated the phosphorylation of tau by activating microsomal PGE synthase 1 (mPGES1) in a prostaglandin (PG) E2-dependent EP receptor-activating manner. Specifically, the highly accumulated Ca2+ stimulated the expression of mPGES1 and the synthesis of PGE2. Treatment with the inhibitor of Ca2+ transporter, NMDAR, attenuated the expression of mPGES1 and the production of PGE2 were attenuated in S(+)-ketamine-treated APP/PS1 Tg mice. Elevated levels of PGE2 were responsible for the hyperphosphorylation of tau in an EP-1-, EP-2-, and EP-3-dependent but not EP4-dependent cyclin-dependent kinase (Cdk) 5-activating manner. Reciprocally, the knockdown of the expression of mPGES1 ameliorated the expected cognitive decline by inhibiting the phosphorylation of tau in APP/PS1 Tg mice. Moreover, CDK5 was found to be located downstream of EP1-3 to regulate the phosphorylation of tau though the cleavage of p35 to p25. Finally, the phosphorylation of tau by Ca2+ contributed to the cognitive decline of APP/PS1 Tg mice.
Project description:Although beta-amyloid (Abeta) plaques and tau neurofibrillary tangles are hallmarks of Alzheimer's disease (AD) neuropathology, loss of synapses is considered the best correlate of cognitive decline in AD, rather than plaques or tangles. How pathological Abeta and tau aggregation relate to each other and to alterations in synapses remains unclear. Since aberrant tau phosphorylation occurs in amyloid precursor protein (APP) Swedish mutant transgenic mice, and since neurofibrillary tangles develop in triple transgenic mice harboring mutations in APP, tau and presenilin 1, we utilized these well-characterized mouse models to explore the relation between Abeta and tau pathologies. We now report that pathological accumulation of Abeta and hyperphosphorylation of tau develop concomitantly within synaptic terminals.
Project description:In the normal brain, tau protein is phosphorylated at a number of proline- and non-proline directed sites, which reduce tau microtubule binding and thus regulate microtubule dynamics. In Alzheimer disease (AD), tau is abnormally hyperphosphorylated, leading to neurofibrillary tangle formation and microtubule disruption, suggesting a loss of regulatory mechanisms controlling tau phosphorylation. Early growth response 1 (Egr-1) is a transcription factor that is significantly up-regulated in AD brain. The pathological significance of this up-regulation is not known. In this study, we found that lentivirus-mediated overexpression of Egr-1 in rat brain hippocampus and primary neurons in culture activates proline-directed kinase Cdk5, inactivates PP1, promotes tau phosphorylation at both proline-directed Ser(396/404) and non-proline-directed Ser(262) sites, and destabilizes microtubules. Furthermore, in Egr-1(-/-) mouse brain, Cdk5 activity was decreased, PP1 activity was increased, and tau phosphorylation was reduced at both proline-directed and non-proline-directed sites. By using nerve growth factor-exposed PC12 cells, we determined that Egr-1 activates Cdk5 to promote phosphorylation of tau and inactivates PP1 via phosphorylation. When Cdk5 was inhibited, tau phosphorylation at both proline- and non-proline directed sites and PP1 phosphorylation were blocked, indicating that Egr-1 acts through Cdk5. By using an in vitro kinase assay and HEK-293 cells transfected with tau, PP1, and Cdk5, we found that Cdk5 phosphorylates Ser(396/404) directly. In addition, by phosphorylating and inactivating PP1, Cdk5 promotes tau phosphorylation at Ser(262) indirectly. Our results indicate that Egr-1 is an in vivo regulator of tau phosphorylation and suggest that in AD brain increased levels of Egr-1 aberrantly activate an Egr-1/Cdk5/PP1 pathway, leading to accumulation of hyperphosphorylated tau, thus destabilizing the microtubule cytoskeleton.
Project description:Alzheimer's disease (AD) neuropathological hallmarks include senile plaques with aggregated amyloid beta as a major component, neurofibrillary tangles (NFT) containing truncated and hyperphosphorylated Tau, extensive neuronal loss, and chronic neuroinflammation. However, the key molecular mechanism that dominates the pathogenesis of AD remains elusive for AD. Here we show that the C/EBPβ/δ-secretase axis is activated in an age-dependent manner in different brain regions of the 3×Tg AD mouse model, elevating δ-secretase-truncated APP and Tau proteolytic truncates and promoting senile plaques and NFT formation in the brain, associated with gradual neuronal loss and chronic neuroinflammation. Depletion of inflammatory cytokine-regulated transcription factor C/EBPβ from 3×Tg mice represses APP, Tau, and δ-secretase expression, which subsequently inhibits APP and Tau cleavage, leading to mitigation of AD pathologies. Knockout of δ-secretase from 3×Tg mice strongly blunts AD pathogenesis. Consequently, inactivation of the C/EBPβ/δ-secretase axis ameliorates cognitive dysfunctions in 3×Tg mice by blocking APP and Tau expression and their pathological fragmentation. Thus, our findings support the notion that C/EBPβ/δ-secretase axis plays a crucial role in AD pathogenesis.
Project description:Alzheimer's disease (AD) is the most common form of dementia and pathologically featured by ?-amyloid (A?) plaque deposition and hyper-phosphorylated tau aggregation in the brain. Environmental factors are believed to contribute to the pathogenesis and progression of AD. In the present study, we investigated the impacts of acute hypoxia on A? and tau pathologies, neuroinflammation, mitochondrial function, and autophagy in APPswe/PS1dE9 AD mouse model. Male APPswe/PS1dE9 transgenic (Tg) mice and their age-matched wild type (Wt) littermates were exposed to one single acute hypoxic episode (oxygen 7%) for 24 h. We found that acute hypoxia exposure increased the expressions of amyloid precursor protein (APP), anterior pharynx-defective 1 (APH1) and cyclin-dependent kinase 5 (CDK5), and promoted tau phosphorylation at T181 and T231 residues in both Tg and Wt mice. In addition, acute hypoxia also induced autophagy through the mammalian target of rapamycin (mTOR) signaling, elicited abnormal mitochondrial function and neuroinflammation in both Tg and Wt mice. In summary, all these findings suggest that acute hypoxia could induce the AD-like pathological damages in the brain of APPswe/PS1dE9 mice and Wt mice to some extent.
Project description:The triple-transgenic Alzheimer (3 × Tg-AD) mouse expresses mutant PS1(M146V), APP(swe), and tau(P301L) transgenes and progressively develops plaques and neurofibrillary tangles with a temporal- and region-specific profile that resembles the neuropathological progression of Alzheimer's disease (AD). In this study, we used proteomic approaches such as two-dimensional gel electrophoresis and mass spectrometry to investigate the alterations in protein expression occurring in the brain and cerebellum of 3 × Tg-AD and presenilin-1 (PS1) knock-in mice (animals that do not develop A?- or tau-dependent pathology nor cognitive decline and were used as control). Finally, using the Ingenuity Pathway Analysis we evaluated novel networks and molecular pathways involved in this AD model. We identified several differentially expressed spots and analysis of 3 × Tg-AD brains showed a significant downregulation of synaptic proteins that are involved in neurotransmitter synthesis, storage and release, as well as a set of proteins that are associated with cytoskeleton assembly and energy metabolism. Interestingly, in the cerebellum, a structure not affected by AD, we found an upregulation of proteins involved in carbohydrate metabolism and protein catabolism. Our findings help to unravel the pathogenic brain mechanisms set in motion by mutant amyloid precursor protein (APP) and hyperphosphorylated tau. These data also reveal cerebellar pathways that may be important to counteract the pathogenic actions of A? and tau, and ultimately offer novel targets for therapeutic intervention.
Project description:Hyperphosphorylation of tau at multiple sites has been implicated in the formation of neurofibrillary tangles in Alzheimer's disease; however, the relationship between toxicity and phosphorylation of tau has not been clearly elucidated. Putative tau kinases that play a role in such phosphorylation events include the proline-directed kinases glycogen synthase kinase-3beta (GSK-3beta) and cyclin-dependent kinase 5 (Cdk5), as well as nonproline-directed kinases such as microtubule affinity-regulating kinase (MARK)/PAR-1; however, whether the cascade of events linking tau phosphorylation and neurodegeneration involves sequential action of kinases as opposed to parallel pathways is still a matter of controversy. Here, we employed a well-characterized Drosophila model of tauopathy to investigate the interdependence of tau kinases in regulating the phosphorylation and toxicity of tau in vivo. We found that tau mutants resistant to phosphorylation by MARK/PAR-1 were indeed less toxic than wild-type tau; however, this was not due to their resistance to phosphorylation by GSK-3beta/Shaggy. On the contrary, a tau mutant resistant to phosphorylation by GSK-3beta/Shaggy retained substantial toxicity and was found to have increased affinity for microtubules compared with wild-type tau. The fly homologs of Cdk5/p35 did not have major effects on tau toxicity or phosphorylation in this model. These data suggest that, in addition to tau phosphorylation, microtubule binding plays a crucial role in the regulation of tau toxicity when misexpressed. These data have important implications for the understanding and interpretation of animal models of tauopathy.
Project description:UNLABELLED:Despite the demonstration that amyloid-? (A?) can trigger increased tau phosphorylation and neurofibrillary tangle (NFT) formation in vivo, the molecular link associating A? and tau pathologies remains ill defined. Here, we observed that exposure of cultured primary neurons to A? trimers isolated from brain tissue of subjects with Alzheimer's disease led to a specific conformational change of tau detected by the antibody Alz50. A similar association was supported by postmortem human brain analyses. To study the role of A? trimers in vivo, we created a novel bigenic Tg-A?+Tau mouse line by crossing Tg2576 (Tg-A?) and rTg4510 (Tg-Tau) mice. Before neurodegeneration and amyloidosis, apparent A? trimers were increased by ?2-fold in 3-month-old Tg-A? and Tg-A?+Tau mice compared with younger mice, whereas soluble monomeric A? levels were unchanged. Under these conditions, the expression of soluble Alz50-tau conformers rose by ?2.2-fold in the forebrains of Tg-A?+Tau mice compared with nontransgenic littermates. In parallel, APP accumulated intracellularly, suggestive of a putative dysfunction of anterograde axonal transport. We found that the protein abundance of the kinesin-1 light chain (KLC1) was reduced selectively in vivo and in vitro when soluble A? trimers/Alz50-tau were present. Importantly, the reduction in KLC1 was prevented by the intraneuronal delivery of Alz50 antibodies. Collectively, our findings reveal that specific soluble conformers of A? and tau cooperatively disrupt axonal transport independently from plaques and tangles. Finally, these results suggest that not all endogenous A? oligomers trigger the same deleterious changes and that the role of each assembly should be considered separately. SIGNIFICANCE STATEMENT:The mechanistic link between amyloid-? (A?) and tau, the two major proteins composing the neuropathological lesions detected in brain tissue of Alzheimer's disease subjects, remains unclear. Here, we report that the trimeric A? species induce a pathological modification of tau in cultured neurons and in bigenic mice expressing A? and human tau. This linkage was also observed in postmortem brain tissue from subjects with mild cognitive impairment, when A? trimers are abundant. Further, this modification of tau was associated with the intracellular accumulation of the precursor protein of A?, APP, as a result of the selective decrease in kinesin light chain 1 expression. Our findings suggest that A? trimers might cause axonal transport deficits in AD.
Project description:Accumulation of A? peptide fragments of the APP protein and neurofibrillary tangles of the microtubule-associated protein tau are the cellular hallmarks of Alzheimer's disease (AD). To investigate the relationship between APP metabolism and tau protein levels and phosphorylation, we studied human-stem-cell-derived forebrain neurons with genetic forms of AD, all of which increase the release of pathogenic A? peptides. We identified marked increases in intracellular tau in genetic forms of AD that either mutated APP or increased its dosage, suggesting that APP metabolism is coupled to changes in tau proteostasis. Manipulating APP metabolism by ?-secretase and ?-secretase inhibition, as well as ?-secretase modulation, results in specific increases and decreases in tau protein levels. These data demonstrate that APP metabolism regulates tau proteostasis and suggest that the relationship between APP processing and tau is not mediated solely through extracellular A? signaling to neurons.