Beta-amyloid treatment of two complementary P301L tau-expressing Alzheimer's disease models reveals similar deregulated cellular processes

Alzheimer's disease (AD) is characterized by Abeta peptide-containing plaques and tau-containing neurofibrillary tangles (NFTs). Both pathologies have been combined by crossing Abeta plaque-forming APP mutant mice with NFT-forming P301L tau mutant mice or by stereotaxically injecting beta-amyloid peptide 1-42 (Abeta42) into brains of P301L tau mutant mice. In cell culture, Abeta42 induces filamentous tau aggregates. To understand which processes are disrupted by Abeta42 in the presence of tau aggregates, we applied comparative proteomics to Abeta42-treated P301L tau-expressing neuroblastoma cells and the amygdala of P301L tau transgenic mice stereotaxically injected with Abeta42. Remarkably, a significant fraction of proteins altered in both systems belonged to the same functional categories, i.e. stress response and metabolism. We also identified model-specific effects of Abeta42 treatment such as differences in cell signaling proteins in the cellular model and of cytoskeletal and synapse associated proteins in the amygdala. By Western blotting (WB) and immunohistochemistry (IHC), we were able to show that 72% of the tested candidates were altered in human AD brain with a major emphasis on stress-related unfolded protein responsive candidates. These data highlight these processes as potentially important initiators in the Abeta42-mediated pathogenic cascade in AD and further support the role of unfolded proteins in the course of AD.     

T-817MA, a neuroprotective agent, attenuates the motor and cognitive impairments associated with neuronal degeneration in P301L tau transgenic mice

Tau pathology is implicated in mechanisms of neurodegenerative tauopathies, including Alzheimer’s disease (AD) and hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). It has been reported that transgenic mice expressing FTDP-17 mutation P301L of human tau (P301L mice) display extensive tau pathology and exhibit behavioral deficits with aging. In this study, we investigated the effects of T-817MA, a neuroprotective agent, on the motor and cognitive impairments associated with neuronal degeneration in P301L mice. T-817MA prevented the progression of motor deficit and the loss of spinal cord motor neurons in P301L mice. Furthermore, T-817MA significantly attenuated the spatial memory impairment and the reduction in synaptic terminal density in the hippocampal dentate gyrus of P301L mice. These results indicate that T-817MA improved the motor and cognitive impairments as a result of inhibiting neuronal degeneration derived from tau pathology in the P301L mice. Therefore, it is expected that T-817MA has a therapeutic potential for tau-related neurodegenerative diseases such as AD.     

A New Link to Mitochondrial Impairment in Tauopathies

Tauopathies like the "frontotemporal dementia with Parkinsonism linked to chromosome 17" (FTDP-17) are characterized by an aberrant accumulation of intracellular neurofibrillary tangles composed of hyperphosphorylated tau. For FTDP-17, a pathogenic tau mutation P301L was identified. Impaired mitochondrial function including disturbed dynamics such as fission and fusion are most likely major pathomechanisms of most neurodegenerative diseases. However, very little is known if tau itself affects mitochondrial function and dynamics. We addressed this question using SY5Y cells stably overexpressing wild-type (wt) and P301L mutant tau. P301L overexpression resulted in a substantial complex I deficit accompanied by decreased ATP levels and increased susceptibility to oxidative stress. This was paralleled by pronounced changes in mitochondrial morphology, decreased fusion and fission rates accompanied by reduced expression of several fission and fusion factors like OPA-1 or DRP-1. In contrast, overexpression of wt tau exhibits protective effects on mitochondrial function and dynamics including enhanced complex I activity. Our findings clearly link tau bidirectional to mitochondrial function and dynamics, identifying a novel aspect of the physiological role of tau and the pathomechanism of tauopathies.     

Convergence of Amyloid-β and Tau Pathologies on Mitochondria In Vivo

The histopathological characteristics of Alzheimer’s disease (AD) are amyloid-β (Aβ) containing plaques and neurofibrillary tangles (NFTs) as well as neuronal and synaptic loss. Until today, the underlying mechanisms of the interplay of plaques and tangles remained unresolved. There is increasing evidence that mitochondrial dysfunction might be a possible link, as revealed by studies in several APP and tau transgenic mouse models. Recently, we examined mitochondrial function in a novel triple transgenic mouse model (pR5/APP/PS2)—^tripleAD mice—that combines both pathologic features of the disease in brain. Using comparative, quantitative proteomics (iTRAQ) and mass spectroscopy, we found a massive deregulation of 24 proteins, of which one third were mitochondrial proteins mainly related to complexes I and IV of the oxidative phosphorylation system (OXPHOS). Remarkably, deregulation of complex I was related to tau, whereas deregulation of complex IV was Aβ dependent, both at the protein and activity levels. The ^tripleAD mice showed synergistic effects of Aβ and tau already at the age of 8 months, resulting in a depolarized mitochondrial membrane potential. At 12 months, the strongest defects on OXPHOS, synthesis of ATP and reactive oxygen species, were exhibited in the ^tripleAD mice, again emphasizing synergistic, age-associated effects of Aβ and tau in impairing mitochondria. This review highlights the convergence of Aβ and tau on mitochondria and establishes a molecular link in AD pathology in vivo.     

Effects of Alzheimer's amyloid-beta and tau protein on mitochondrial function -- role of glucose metabolism and insulin signalling

Alzheimer's disease (AD) is the most frequent form of dementia among the elderly and is characterized by neuropathological hallmarks of extracellular amyloid-beta (Abeta) plaques and intracellular neurofibrillary tangles composed of abnormally hyperphosphorylated microtubular protein tau in the brains of AD patients. Of note, current data illustrate a complex interplay between the amyloid and tau pathology during the course of the disease. We hypothesize a direct impact of abnormally phosphorylated tau and Abeta on proteins/enzymes involved in metabolism, respiratory chain function and cellular detoxification. Probably at the level of mitochondria, both Alzheimer proteins exhibit synergistic effects finally leading to/accelerating neurodegenerative mechanisms. Moreover, accumulating evidence that mitochondria failure, reduced glucose utilization and deficient energy metabolism occur already very early in the course of the disease suggests a role of impaired insulin signalling in the pathogenesis of AD. Thus, this review addresses also the question if mitochondrial dysfunction may represent a link between diabetes and AD.     

Aging sensitizes toward ROS formation and lipid peroxidation in PS1M146L transgenic mice

Mutations in the presenilins (PS) account for the majority of familial Alzheimer disease (FAD) cases. To test the hypothesis that oxidative stress can underlie the deleterious effects of presenilin mutations, we analyzed lipid peroxidation products (4-hydroxynonenal (HNE) and malondialdehyde) and antioxidant defenses in brain tissue and levels of reactive oxygen species (ROS) in splenic lymphocytes from transgenic mice bearing human PS1 with the M146L mutation (PS1M146L) compared to those from mice transgenic for wild-type human PS1 (PS1wt) and nontransgenic littermate control mice. In brain tissue, HNE levels were increased only in aged (19-22 months) PS1M146L transgenic animals compared to PS1wt mice and not in young (3-4 months) or middle-aged mice (13-15 months). Similarly, in splenic lymphocytes expressing the transgenic PS1 proteins, mitochondrial and cytosolic ROS levels were elevated to 142.1 and 120.5% relative to controls only in cells from aged PS1M146L animals. Additionally, brain tissue HNE levels were positively correlated with mitochondrial ROS levels in splenic lymphocytes, indicating that oxidative stress can be detected in different tissues of PS1 transgenic mice. Antioxidant defenses (activities of antioxidant enzymes Cu/Zn-SOD, GPx, or GR) or susceptibility to in vitro oxidative stimulation was unaltered. In summary, these results demonstrate that the PS1M146L mutation increases mitochondrial ROS formation and oxidative damage in aged mice. Hence, oxidative stress caused by the combined effects of aging and PS1 mutations may be causative for triggering neurodegenerative events in FAD patients.     

Mitochondrial oxidative stress causes hyperphosphorylation of tau

Age-related neurodegenerative disease has been mechanistically linked with mitochondrial dysfunction via damage from reactive oxygen species produced within the cell. We determined whether increased mitochondrial oxidative stress could modulate or regulate two of the key neurochemical hallmarks of Alzheimer's disease (AD): tau phosphorylation, and beta-amyloid deposition. Mice lacking superoxide dismutase 2 (SOD2) die within the first week of life, and develop a complex heterogeneous phenotype arising from mitochondrial dysfunction and oxidative stress. Treatment of these mice with catalytic antioxidants increases their lifespan and rescues the peripheral phenotypes, while uncovering central nervous system pathology. We examined sod2 null mice differentially treated with high and low doses of a catalytic antioxidant and observed striking elevations in the levels of tau phosphorylation (at Ser-396 and other phospho-epitopes of tau) in the low-dose antioxidant treated mice at AD-associated residues. This hyperphosphorylation of tau was prevented with an increased dose of the antioxidant, previously reported to be sufficient to prevent neuropathology. We then genetically combined a well-characterized mouse model of AD (Tg2576) with heterozygous sod2 knockout mice to study the interactions between mitochondrial oxidative stress and cerebral Ass load. We found that mitochondrial SOD2 deficiency exacerbates amyloid burden and significantly reduces metal levels in the brain, while increasing levels of Ser-396 phosphorylated tau. These findings mechanistically link mitochondrial oxidative stress with the pathological features of AD.     

Reduction of soluble Abeta and tau, but not soluble Abeta alone, ameliorates cognitive decline in transgenic mice with plaques and tangles

Increasing evidence points to soluble assemblies of aggregating proteins as a major mediator of neuronal and synaptic dysfunction. In Alzheimer disease (AD), soluble amyloid-beta (Abeta) appears to be a key factor in inducing synaptic and cognitive abnormalities. Here we report the novel finding that soluble tau also plays a role in the cognitive decline in the presence of concomitant Abeta pathology. We describe improved cognitive function following a reduction in both soluble Abeta and tau levels after active or passive immunization in advanced aged 3xTg-AD mice that contain both amyloid plaques and neurofibrillary tangles (NFTs). Notably, reducing soluble Abeta alone did not improve the cognitive phenotype in mice with plaques and NFTs. Our results show that Abeta immunotherapy reduces soluble tau and ameliorates behavioral deficit in old transgenic mice.     

Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction

The neuropathological correlates of Alzheimer's disease (AD) include amyloid-beta (Abeta) plaques and neurofibrillary tangles. To study the interaction between Abeta and tau and their effect on synaptic function, we derived a triple-transgenic model (3xTg-AD) harboring PS1(M146V), APP(Swe), and tau(P301L) transgenes. Rather than crossing independent lines, we microinjected two transgenes into single-cell embryos from homozygous PS1(M146V) knockin mice, generating mice with the same genetic background. 3xTg-AD mice progressively develop plaques and tangles. Synaptic dysfunction, including LTP deficits, manifests in an age-related manner, but before plaque and tangle pathology. Deficits in long-term synaptic plasticity correlate with the accumulation of intraneuronal Abeta. These studies suggest a novel pathogenic role for intraneuronal Abeta with regards to synaptic plasticity. The recapitulation of salient features of AD in these mice clarifies the relationships between Abeta, synaptic dysfunction, and tangles and provides a valuable model for evaluating potential AD therapeutics as the impact on both lesions can be assessed.     

Proteomic analysis of specific brain proteins in aged SAMP8 mice treated with alpha-lipoic acid: implications for aging and age-related neurodegenerative disorders

Free radical-mediated damage to neuronal membrane components has been implicated in the etiology of Alzheimer's disease (AD) and aging. The senescence accelerated prone mouse strain 8 (SAMP8) exhibits age-related deterioration in memory and learning along with increased oxidative markers. Therefore, SAMP8 is a suitable model to study brain aging and, since aging is the major risk factor for AD and SAMP8 exhibits many of the biochemical findings of AD, perhaps as a model for and the early phase of AD. Our previous studies reported higher oxidative stress markers in brains of 12-month-old SAMP8 mice when compared to that of 4-month-old SAMP8 mice. Further, we have previously shown that injecting the mice with alpha-lipoic acid (LA) reversed brain lipid peroxidation, protein oxidation, as well as the learning and memory impairments in SAMP8 mice. Recently, we reported the use of proteomics to identify proteins that are expressed differently and/or modified oxidatively in aged SAMP8 brains. In order to understand how LA reverses the learning and memory deficits of aged SAMP8 mice, in the current study, we used proteomics to compare the expression levels and specific carbonyl levels of proteins in brains from 12-month-old SAMP8 mice treated or not treated with LA. We found that the expressions of the three brain proteins (neurofilament triplet L protein, alpha-enolase, and ubiquitous mitochondrial creatine kinase) were increased significantly and that the specific carbonyl levels of the three brain proteins (lactate dehydrogenase B, dihydropyrimidinase-like protein 2, and alpha-enolase) were significantly decreased in the aged SAMP8 mice treated with LA. These findings suggest that the improved learning and memory observed in LA-injected SAMP8 mice may be related to the restoration of the normal condition of specific proteins in aged SAMP8 mouse brain. Moreover, our current study implicates neurofilament triplet L protein, alpha-enolase, ubiquitous mitochondrial creatine kinase, lactate dehydrogenase B, and dihydropyrimidinase-like protein 2 in process associated with learning and memory of SAMP8 mice.     

Glutathione peroxidase‐1 overexpression reduces oxidative stress,and improves pathology and proteome remodeling in the kidneys of old mice

This study investigated the direct roles of hydrogen peroxide (H₂O₂) in kidney aging using transgenic mice overexpressing glutathione peroxidase‐1 (GPX1 TG). We demonstrated that kidneys in old mice recapitulated kidneys in elderly humans and were characterized by glomerulosclerosis, tubular atrophy, interstitial fibrosis, and loss of cortical mass. Scavenging H₂O₂ by GPX1 TG significantly reduced mitochondrial and total cellular reactive oxygen species (ROS) and mitigated oxidative damage, thus improving these pathologies. The potential mechanisms by which ROS are increased in the aged kidney include a decreased abundance of an anti‐aging hormone, Klotho, in kidney tissue, and decreased expression of nuclear respiratory factor 2 (Nrf2), a master regulator of the stress response. Decreased Klotho or Nrf2 was not improved in the kidneys of old GPX1 TG mice, even though mitochondrial morphology was better preserved. Using laser capture microdissection followed by label‐free shotgun proteomics analysis, we show that the glomerular proteome in old mice was characterized by decreased abundance of cytoskeletal proteins (critical for maintaining normal glomerular function) and heat shock proteins, leading to increased accumulation of apolipoprotein E and inflammatory molecules. Targeted proteomic analysis of kidney tubules from old mice showed decreased abundance of fatty acid oxidation enzymes and antioxidant proteins, as well as increased abundance of glycolytic enzymes and molecular chaperones. GPX1 TG partially attenuated the remodeling of glomerular and tubule proteomes in aged kidneys. In summary, mitochondria from GPX1 TG mice are protected and kidney aging is ameliorated via its antioxidant activities, independent and downstream of Nrf2 or Klotho signaling.     

Changes in mitochondrial glutathione levels and protein thiol oxidation in ∆yfh1 yeast cells and the lymphoblasts of patients with Friedreich's ataxia

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by low levels of the mitochondrial protein frataxin. The main phenotypic features of frataxin-deficient human and yeast cells include iron accumulation in mitochondria, iron-sulfur cluster defects and high sensitivity to oxidative stress. Frataxin deficiency is also associated with severe impairment of glutathione homeostasis and changes in glutathione-dependent antioxidant defenses. The potential biological consequences of oxidative stress and changes in glutathione levels associated with frataxin deficiency include the oxidation of susceptible protein thiols and reversible binding of glutathione to the SH of proteins by S-glutathionylation. In this study, we isolated mitochondria from frataxin-deficient ?yfh1 yeast cells and lymphoblasts of FRDA patients, and show evidence for a severe mitochondrial glutathione-dependent oxidative stress, with a low GSH/GSSG ratio, and thiol modifications of key mitochondrial enzymes. Both yeast and human frataxin-deficient cells had abnormally high levels of mitochondrial proteins binding an anti-glutathione antibody. Moreover, proteomics and immunodetection experiments provided evidence of thiol oxidation in α-ketoglutarate dehydrogenase (KGDH) or subunits of respiratory chain complexes III and IV. We also found dramatic changes in GSH/GSSG ratio and thiol modifications on aconitase and KGDH in the lymphoblasts of FRDA patients. Our data for yeast cells also confirm the existence of a signaling and/or regulatory process involving both iron and glutathione.     

Increasing Brain Protein O-GlcNAc-ylation Mitigates Breathing Defects and Mortality of Tau.P301L Mice

The microtubule associated protein tau causes primary and secondary tauopathies by unknown molecular mechanisms. Post-translational O-GlcNAc-ylation of brain proteins was demonstrated here to be beneficial for Tau.P301L mice by pharmacological inhibition of O-GlcNAc-ase. Chronic treatment of ageing Tau.P301L mice mitigated their loss in body-weight and improved their motor deficits, while the survival was 3-fold higher at the pre-fixed study endpoint at age 9.5 months. Moreover, O-GlcNAc-ase inhibition significantly improved the breathing parameters of Tau.P301L mice, which underpinned pharmacologically the close correlation of mortality and upper-airway defects. O-GlcNAc-ylation of brain proteins increased rapidly and stably by systemic inhibition of O-GlcNAc-ase. Conversely, biochemical evidence for protein Tau.P301L to become O-GlcNAc-ylated was not obtained, nor was its phosphorylation consistently or markedly affected. We conclude that increasing O-GlcNAc-ylation of brain proteins improved the clinical condition and prolonged the survival of ageing Tau.P301L mice, but not by direct biochemical action on protein tau. The pharmacological effect is proposed to be located downstream in the pathological cascade initiated by protein Tau.P301L, opening novel venues for our understanding, and eventually treating the neurodegeneration mediated by protein tau.     

Effect of DL-alpha-lipoic acid on mitochondrial enzymes in aged rats.

Mitochondrial dysfunction appears to contribute to some of the loss of function accompanying ageing. Mitochondria from aged tissue use oxygen inefficiently impairing ATP synthesis and results in increased oxidant production. A high flux of oxidants not only damages mitochondria, but other important cell biomolecules as well. In the present investigation, the levels of lipid peroxidation, oxidized glutathione, non-enzymatic antioxidants and the activities of mitochondrial enzymes were measured in liver and kidney mitochondria of young and aged rats before and after lipoic acid supplementation. In both liver and kidney increase in the levels of mitochondrial lipid peroxidation and oxidized glutathione and decrease in the levels of antioxidants and the activities of mitochondrial enzymes were observed in aged rats. DL-alpha-lipoic acid supplemented aged rats showed a decrease in the levels of lipid peroxidation and oxidized glutathione and increase in the levels of reduced glutathione, vitamins C and E and the activities of mitochondrial enzymes like isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, NADH-dehydrogenase and cytochrome-c-oxidase. Thus, lipoic acid reverses the age-associated decline in endogenous low molecular weight antioxidants and mitochondrial enzymes and, therefore, may lower the increased risk of oxidative damage that occurs during ageing. From our results it can be concluded that lipoic acid supplementation enhances the activities of mitochondrial enzymes and antioxidant status and thereby protects mitochondria from ageing.     

Genotype-specific differences between mouse CNS stem cell lines expressing frontotemporal dementia mutant or wild type human tau

Stem cell (SC) lines that capture the genetics of disease susceptibility provide new research tools. To assess the utility of mouse central nervous system (CNS) SC-containing neurosphere cultures for studying heritable neurodegenerative disease, we compared neurosphere cultures from transgenic mice that express human tau with the P301L familial frontotemporal dementia (FTD) mutation, rTg(tau(P301L))4510, with those expressing comparable levels of wild type human tau, rTg(tau(wt))21221. rTg(tau(P301L))4510 mice express the human tau(P301L) variant in their forebrains and display cellular, histological, biochemical and behavioral abnormalities similar to those in human FTD, including age-dependent differences in tau phosphorylation that distinguish them from rTg(tau(wt))21221 mice. We compared FTD-hallmark tau phosphorylation in neurospheres from rTg(tau(P301L))4510 mice and from rTg(tau(wt))21221 mice. The tau genotype-specific phosphorylation patterns in neurospheres mimicked those seen in mice, validating use of neurosphere cultures as models for studying tau phosphorylation. Genotype-specific tau phosphorylation was observed in 35 independent cell lines from individual fetuses; tau in rTg(tau(P301L))4510 cultures was hypophosphorylated in comparison with rTg(tau(wt))21221 as was seen in young adult mice. In addition, there were fewer human tau-expressing cells in rTg(tau(P301L))4510 than in rTg(tau(wt))21221 cultures. Following differentiation, neuronal filopodia-spine density was slightly greater in rTg(tau(P301L))4510 than rTg(tau(wt))21221 and control cultures. Together with the recapitulation of genotype-specific phosphorylation patterns, the observation that neurosphere lines maintained their cell line-specific-differences and retained SC characteristics over several passages supports the utility of SC cultures as surrogates for analysis of cellular disease mechanisms.     

Increased plaque burden in brains of APP mutant MnSOD heterozygous knockout mice

A growing body of evidence suggests a relationship between oxidative stress and beta-amyloid (Abeta) peptide accumulation, a hallmark in the pathogenesis of Alzheimer's disease (AD). However, a direct causal relationship between oxidative stress and Abeta pathology has not been established in vivo. Therefore, we crossed mice with a knockout of one allele of manganese superoxide dismutase (MnSOD), a critical antioxidant enzyme, with Tg19959 mice, which overexpress a doubly mutated human beta-amyloid precursor protein (APP). Partial deficiency of MnSOD, which is well established to cause elevated oxidative stress, significantly increased brain Abeta levels and Abeta plaque burden in Tg19959 mice. These results indicate that oxidative stress can promote the pathogenesis of AD and further support the feasibility of antioxidant approaches for AD therapy.     

Piericidin A Aggravates Tau Pathology in P301S Transgenic Mice

ObjectiveThe P301S mutation in exon 10 of the tau gene causes a hereditary tauopathy. While mitochondrial complex I inhibition has been linked to sporadic tauopathies. Piericidin A is a prototypical member of the group of the piericidins, a class of biologically active natural complex I inhibitors, isolated from streptomyces spp. with global distribution in marine and agricultural habitats. The aim of this study was to determine whether there is a pathogenic interaction of the environmental toxin piericidin A and the P301S mutation.MethodsTransgenic mice expressing human tau with the P301S-mutation (P301S^+/+) and wild-type mice at 12 weeks of age were treated subcutaneously with vehicle (N = 10 P301S^+/+, N = 7 wild-type) or piericidin A (N = 9 P301S^+/+, N = 9 wild-type mice) at a dose of 0.5 mg/kg/d for a period of 28 days via osmotic minipumps. Tau pathology was measured by stereological counts of cells immunoreative with antibodies against phosphorylated tau (AD2, AT8, AT180, and AT100) and corresponding Western blot analysis.ResultsPiericidin A significantly increased the number of phospho-tau immunoreactive cells in the cerebral cortex in P301S^+/+ mice, but only to a variable and mild extent in wild-type mice. Furthermore, piericidin A led to increased levels of pathologically phosphorylated tau only in P301S^+/+ mice. While we observed no apparent cell loss in the frontal cortex, the synaptic density was reduced by piericidin A treatment in P301S^+/+ mice.DiscussionThis study shows that exposure to piericidin A aggravates the course of genetically determined tau pathology, providing experimental support for the concept of gene-environment interaction in the etiology of tauopathies.     

Changed conformation of mutant Tau-P301L underlies the moribund tauopathy, absent in progressive, nonlethal axonopathy of Tau-4R/2N transgenic mice

Protein tau-3R/4R isoform ratio and phosphorylation regulates binding to microtubules and, when disturbed by aging or mutations, results in diverse tauopathies and in neurodegeneration. The underlying mechanisms were studied here in three transgenic mouse strains with identical genetic background, all expressing the tau-4R/2N isoform driven specifically in neurons by the thy1 gene promoter. Two strains, expressing human tau-4R/2N or mutant tau-4R/2N-P301L at similar, moderate levels, developed very different phenotypes. Tau-4R/2N mice became motor-impaired already around age 6-8 weeks, accompanied by axonopathy (dilatations, spheroids), but no tau aggregates, and surviving normally. In contrast, tau-P301L mice developed neurofibrillary tangles from age 6 months, without axonal dilatations and, despite only minor motor problems, all succumbing before the age of 13 months. The third strain, obtained by tau knock-out/knock-in (tau-KOKI), expressed normal levels of wild-type human tau-4R/2N replacing all mouse tau isoforms. Tau-KOKI mice survived normally with minor motor problems late in life and without any obvious pathology. Biochemically, a fraction of neuronal tau in aging tau-P301L mice was hyperphosphorylated concomitant with conformational changes and aggregation, but overall, tau-4R/2N was actually more phosphorylated than tau-P301L. Significantly, tau with changed conformation and with hyperphosphorylation colocalized in the same neurons in aging tau-P301L mice. Taken together, we conclude that excessive binding of tau-4R/2N as opposed to reduced binding of tau-P301L to microtubules is responsible for the development of axonopathy and tauopathy, respectively, in tau-4R/2N and tau-P301L mice and that the conformational change of tau-P301L is a major determinant in triggering the tauopathy.