Tau and axonopathy in neurodegenerative disorders

The microtubule (MT)-associated protein (MAP) tau in neurons has been implicated as a significant factor in the axonal growth, development of neuronal polarity, and the maintenance of MT dynamics. Tau is localized to the axon, and is known to promote MT assembly and to stabilize axonal MTs. These functions of tau are primarily regulated by the activities of protein kinases and phosphatases. In Alzheimer's disease and other neurodegenerative disorders, abundant filamentous tau inclusions are found to be major neuropathological characteristics of these diseases. Both somato-dendritic and axonal tau lesions appear to be closely associated with axonal disruption. Furthermore, recent discoveries of pathogenic mutations on the tau gene suggest that abnormalities of tau alone are causative of neurodegeneration. Finally, analyses of transgenic mice that express human tau proteins have enabled in vivo quantitative assessments of axonal functions and have provided information about mechanistic relationships between pathological alteration of tau and axonal degeneration.     

Long-term oral lithium treatment attenuates motor disturbance in tauopathy model mice: implications of autophagy promotion

Lithium, a drug used to treat bipolar disorders, has a variety of neuroprotective mechanisms including inhibition of glycogen synthase kinase-3 (GSK-3), a major tau kinase. Recently, it has been shown that, in various neurodegenerative proteinopathies, lithium could induce autophagy. To analyze how lithium is therapeutically beneficial in tauopathies, transgenic mice overexpressing human mutant tau (P301L) were treated with oral lithium chloride (LiCl) for 4 months starting at the age of 5 months. At first, we examined the effects of treatment on behavior (using a battery of behavioral tests), tau phosphorylation (by biochemical assays), and number of neurofibrillary tangles (NFTs) (by immunohistopathology). In comparison with control mice, LiCl-treated mice showed a significantly better score in the sensory motor tasks, as well as decreases in tau phosphorylation, soluble tau level, and number of NFTs. Next, we examined lithium effects on autophagy using an antibody against microtubule-associated protein 1 light chain 3 (LC3) as an autophagosome marker. The number of LC3-positive autophagosome-like puncta was increased in neurons of LiCl-treated mice. Neurons containing NFTs were completely LC3-negative, whereas LC3-positive autophagosome-like puncta contained phosphorylated-tau (p-tau). The protein level of p62 was decreased in LiCl-treated mice. These data suggested that oral long-term lithium treatment could attenuate p-tau-induced motor disturbance not only by inhibiting GSK-3 but also by enhancing autophagy in tauopathy model mice.     

Tau oligomers as potential targets for immunotherapy for Alzheimer's disease and tauopathies

The aggregation and accumulation of the microtubule-associated protein (Tau) is a pathological hallmark of Alzheimer disease (AD) and many neurodegenerative diseases. For a long time research has focused on neurofibrillary tangles (NFTs) and other large meta-stable inclusions composed of aggregated hyperphosphorylated tau protein. The correlation between these structures and disease progression produced conflicting results; moreover, the mechanism of their formation remains poorly understood. Lately, the significance and toxicity of NFTs have been challenged and a new aggregated tau entity has emerged as the true pathogenic species in tauopathies and a possible mediator of Aβ toxicity in AD; specifically, aggregates of a size intermediate between monomers and NFTs the so-called tau oligomers. Tremendous efforts have been devoted toward the optimization of a safe vaccine for AD by targeting Aβ peptide; despite the disappointing results, these studies produced a wealth of useful knowledge, which should be considered in developing tau-based immunotherapy. Herein, we discuss the evidence supporting the critical role of tau oligomers in AD, the potential and challenges for targeting them by immunotherapy as a novel approach for AD treatment.     

Tau in neurodegenerative diseases: Tau phosphorylation and assembly

The possible link between tau phosphorylation and tau assembly in these neurodegenerative diseases known as tauopathies is described. Additionally, this link is supported by an in vitro experiment showing the higher capacity of phosphotau to assemble in some specific conditions; and, by a recently reported experiment using a tau transgenic mouse model.     

Neurofibrillary tangle formation by introducing wild-type human tau into APP transgenic mice

Senile plaques comprised of Aβ aggregates and neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau filaments are the hallmarks of Alzheimer’s disease (AD). A number of amyloid precursor protein (APP) transgenic (Tg) mice harboring APP mutations have been generated as animal models of AD. These mice successfully display amyloid plaque formation and subsequent tau hyperphosphorylation, but seldom induce NFT formations. We have demonstrated that the APP_OSK-Tg mice, which possess the E693Δ (Osaka) mutation in APP and thereby accumulate Aβ oligomers without plaques, exhibit tau hyperphosphorylation at 8 months, but not NFT formation even at 24 months. We assumed that APP-Tg mice, including ours, failed to form NFTs because NFT formation requires human tau. To test this hypothesis, we crossbred APP_OSK-Tg mice with tau-Tg mice (tau264), which express low levels of 3-repeat and 4-repeat wild-type human tau without any pathology. The resultant double Tg mice displayed tau hyperphosphorylation at 6 months and NFT formation at 18 months in the absence of tau mutations. Importantly, these NFTs contained both 3-repeat and 4-repeat human tau, similar to those in AD. Furthermore, the double Tg mice exhibited Aβ oligomer accumulation, synapse loss, and memory impairment at 6 months and neuronal loss at 18 months, all of which appeared earlier than in the parent APP_OSK-Tg mice. These results suggest that Aβ and human tau synergistically interact to accelerate each other’s pathology, that the presence of human tau is critical for NFT formation, and that Aβ oligomers can induce NFTs in the absence of amyloid plaques.     

Proteomic analysis of plasma from a Tau transgenic mouse

The neurofibrillary tangles (NFTs) formed by the accumulation of abnormal tau filaments have been shown to be involved in Alzheimer's disease (AD) brain degeneration. In this study, a tau transgenic mouse (pNSE/htau23) model was used to monitor changes in protein levels and to search for novel biomarker candidates suitable for the early diagnosis of AD before onset of clinical symptoms. Plasma samples from 2-month (n=13, asymptomatic) and 4-month (n=7, symptomatic) tau transgenic mice were compared to the control group (n=8) by 2-dimensional gel electrophoresis (2-DE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Three proteins, ATP synthase, Adenosine kinase and Regucalcin showed significantly decreased levels in the plasma of tau transgenic mouse, which was further confirmed by Western blotting. This study suggests that these proteins could be used as candidate biomarkers for early diagnosis of AD in combination with previously discovered protein biomarkers.     

Tau isoform regulation is region- and cell-specific in mouse brain

Tau is a microtubule-associated protein implicated in neurodegenerative tauopathies. Alternative splicing of the tau gene (MAPT) generates six tau isoforms, distinguishable by the exclusion or inclusion of a repeat region of exon 10, which are referred to as 3-repeat (3R) and 4-repeat (4R) tau, respectively. We developed transgenic mouse models that express the entire human MAPT gene in the presence and absence of the mouse Mapt gene and compared the expression and regulation of mouse and human tau isoforms during development and in the young adult. We found differences between mouse and human tau in the regulation of exon 10 inclusion. Despite these differences, the isoform splicing pattern seen in normal human brain is replicated in our mouse models. In addition, we found that all tau, both in the neonate and young adult, is phosphorylated. We also examined the normal anatomic distribution of mouse and human tau isoforms in mouse brain. We observed developmental and species-specific variations in the expression of 3R- and 4R-tau within the frontal cortex and hippocampus. In addition, there were differences in the cellular distribution of the isoforms. Mice transgenic for the human MAPT gene exhibited higher levels of neuronal cell body expression of tau compared to wildtype mice. This neuronal cell body expression of tau was limited to the 3R isoform, whereas expression of 4R-tau was more "synaptic like," with granular staining of neuropil rather than in neuronal cell bodies. These developmental and species-specific differences in the regulation and distribution of tau isoforms may be important to the understanding of normal and pathologic tau isoform expression.     

神经系统退行性疾病相关的tau蛋白及其转基因动物模型

Tau蛋白是存神经系统发育中具有重要生理作用的人脑内含量最高的微管相关蛋白．其功能异常可引起多种神经系统退行性改变。本文就tau蛋白的结构和功能及与神经系统退行性疾病的关系进行了综述，并重点介绍了目前所建立的各种tau蛋白转基因动物模型。     

Tau protein in familial and sporadic diseases

Abnormal protein aggregation is a common characteristic of many neurodegenerative diseases of the brain. Filamentous deposits made of the microtubule-associated protein tau constitute a major defining characteristic of several neurodegenerative diseases known as tauopathies. The role of tau in neurodegeneration has been clarified by the identification of genetic mutations in the tau gene in cases with familial frontotemporal dementia and parkinsonism linked to chromosome 17. Furthermore, some sporadic tauopathies are associated with tau gene polymorphisms. Although it is still debated how tau gene mutations lead to neuronal death, it is clear that different mutations lead to tau pathologies with characteristics similar to those found in sporadic tauopathies. These findings have definitely shown that in tauopathies tau aggregation is directly associated with development of neurodegeneration and neuronal death.     

Analysis of mouse model exhibiting neurofibrillary changes]

Dysfunction and filamentous microtubule-binding tau protein are key markers of neurodegenerative pathologies, including the pathology and neural degeneration associated with Alzheimer's disease (AD). Immunocytochemical studies of NFT-bearing neurons showed that NFTs are composed of ubiquitin and phosphorylation-dependent tau. Congo-red birefringency and thioflavin-S reactivity in NFT-bearing neurons also demonstrated that the tau aggregation forms a beta-sheet structure. Discovery of the molecular mechanisms of NFT formation may lead to more insight about events occurring during neurodegeneration. In frontotemporal dementia parkinsonism 17 (FTDP17), genetic studies indicated that tau is a causative gene, and mutation is found in exons and introns of tau gene. A patient who possesses this mutation exhibits pathologically NFT and clinically personality change and cognitive dysfunction. Then, we produced the Tg mice expressing human longest tau with missense mutation V337M. In the present study, neurons of hippocampus and cerebral cortex in our Tg mice showed phosphorylated and ubiquitinated tau aggregations with a beta-sheet structure. This was demonstrated by Congo-red and thioflavin-S positive staining, a histological criterion used to identify NFTs observed in neurodegenerative disorders. The mice also displayed altered behaviors that were associated with NFT formation. Thus, V337M mice provide a first animal model exhibiting altered behavior due to NFTs.     

Tau and tauopathies

Tau protein is a neuronal microtubule-associated protein (MAP), which localizes primarily in the axon. It is one of the major and most widely distributed MAPs in the central nervous system. Its biochemistry and molecular pathology is being increasingly studied. Tau is a key component of neurofbrillary tangles in Alzheimer's disease (AD). Disorders with neuronal, oligodendroglial or astrocytic filamentous tau inclusions are now grouped under the common rubric of tauopathies. The discovery of mutations in the tau gene, located on Chromosome 17 and its relationship to frontotemporal dementia with Parkinsonism (FTDP-17) has enhanced the importance of tau protein in cognitive neurology. Aberrant aggregates of tau have been documented in most of the neurodegenerative diseases with filamentous inclusions. The role of cerebrospinal fluid tau in the diagnosis of dementias is being investigated quite extensively. Recently, it has been shown that Abeta immunotherapy leads to the clearance of early tau pathology. It is becoming clearer that understanding tau better will lead to better understanding of many neurodegenerative diseases that may help develop interventional strategies.     

RETRACTED ARTICLE: Deferiprone Treatment in Aged Transgenic Tau Mice Improves Y-Maze Performance and Alters Tau Pathology

The accumulation of neurofibrillary tangles (NFTs), which is composed of abnormally hyperphosphorylated tau aggregates, is the classic neuropathology associated with cognitive dysfunction in tauopathies such as Alzheimer’s disease (AD). However, there is an emerging theory suggesting that dysregulation in cerebral iron may contribute to NFT formation. Iron is speculated to bind to tau and induce conformational changes of the protein, potentially leading to subsequent aggregation and cognitive decline. Deferiprone (DFP) is a clinically available iron chelator, which has demonstrated potential therapeutic advantages of chelating iron in neurodegenerative disorders, and is currently in clinical trials for AD. However, its effect on tau pathology remains unclear. Here, we report the effects of short-term DFP treatment (4 weeks, 100 mg/kg/daily, via oral gavage) in a mixed-gender cohort of the rTg_(tauP301L)4510 mouse model of tauopathy. Our results revealed that DFP improved Y-maze and open field performance, accompanied by a 28% decrease in brain iron levels, measured by inductively coupled plasma mass spectrometry (ICP-MS) and reduced AT8-labeled p-tau within the hippocampus in transgenic tau mice. This data supports the notion that iron may play a neurotoxic role in tauopathies and may be a potential therapeutic target for this class of disorders that can be modulated by the clinically available metal chelator DFP.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-020-00972-w.Key Words: Tau, iron, deferiprone, tauopathies, therapeutic     

Novel drugs affecting tau behavior in the treatment of Alzheimer's disease and tauopathies

The anomalous aggregation of proteins into pathological filaments is a common feature of a many human diseases, often related to aging. In this context, neurodegenerative pathologies such as Alzheimer's disease (AD) account for a major part of these protein misfolding diseases. AD is characterized by pathological aggregation of two proteins, tau and Aβ-amyloid. The intracellular neurofibrillary tangles (NFTs) and neuropil threads consists of filaments of the modified microtubule-associated protein tau, while extracellular amyloid plaques consists of filaments of Aβ-peptide. It is noteworthy that tau oligomers with a prefilamentous structure appear to play a role at early stages of AD and tauopathies, but also in asymptomatic patients with Braak-stage I neuropathology, where clinical symptoms of AD and NFTs in frontal cortex are absent. This suggests that an increase in tau oligomers levels occurs before individuals manifest clinical symptoms of AD. NFTs are one of the hallmarks of Alzheimer disease and other tauphaties. These aggregates are thought to be toxic to neurons, either by causing some neurotoxic signalling defects or by obstructing the cell function. Factors contributing to accumulation of tau aggregates include the increased rate of protein misfolding, generation of amyloidogenic oligomers, underactivity of repair systems such as chaperones and ubiquitin-proteasome system, or a failure of energy supply and antioxidant defense mechanisms. There is not clear evidence if the aggregated tau or oligomers cause cellular damage, but on the basis of the emergent need to have an early and effective treatment, lowering the production or removal of these aggregates appears as a pathway toward alleviating the disease. In the context of some of most relevant reports, we analyze why tau protein seems to be an interesting target for AD treatment, and the importance to understand the pathways of tau. aggregation. This knowledge will allow us to identify and optimize potential inhibitors that interact with aggregated forms of tau and hyperphosphorylated tau before the formation of the NFTs, offering a possible therapeutic route for AD treatment.     

Tau and transgenic animal models

Advances in genetics and transgenic approaches have a continuous impact on our understanding of Alzheimer’s disease (AD) and related disorders, especially as aspects of the histopathology and neurodegeneration can be reproduced in animal models. AD is characterized by extracellular Aβ peptide-containing plaques and neurofibrillary aggregates of hyperphosphorylated isoforms of microtubule-associated protein tau. A causal link between Aβ production, neurodegeneration and dementia has been established with the identification of familial forms of AD which are linked to mutations in the amyloid precursor protein APP, from which the Aβ peptide is derived by proteolysis. No mutations have been identified in the tau gene in AD until today. Tau filament formation, in the absence of Aβ production, is also a feature of several additional neurodegenerative diseases including progressive supranuclear palsy, corticobasal degeneration, Pick’s disease, and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). The identification of mutations in the tau gene which are linked to FTDP-17 established that dysfunction of tau can, as well as Aβ formation, lead to neurodegeneration and dementia. In this review, newly recognized cellular functions of tau, and the neuropathology and clinical syndrome of FTDP-17 will be presented, as well as recent advances that have been achieved in studies of transgenic mice expressing tau and AD-related kinases and phosphatases. These models link neurofibrillary lesion formation to neuronal loss, provide an in vivo model in which therapies can be assessed, and may contribute to determine the relationship between Aβ production and tau pathology.     

Amyloid precursor protein cytoplasmic domain with phospho-Thr668 accumulates in Alzheimer’s disease and its transgenic models: a role to mediate interaction of Aβ and tau

Abnormal accumulation of Aβ and tau in senile plaques (SP) and neurofibrillary tangles (NFTs) is a key event in Alzheimer’s disease (AD). Here, we show that T668-phosphorylated cytoplasmic domain of APP (pT668-ACD) accumulates Aβ and tau in AD and its transgenic models. Anti-pT668 immunostaining of AD brain sections with hydrated autoclave enhancement identified SP neurites and NFTs in which pT668-ACD colocalizes with tau. We produced and examined transgenic (Tg) mice that overexpress human APP695, harboring the double Swedish/London mutation, and develop age-dependently Aβ plaques in the brain. All Aβ plaques contain co-accumulations of pT668-ACD, but co-accumulation of tau appears in only a fraction of Aβ plaques in older animals. We also examined the established tau Tg mice that overexpress the smallest human brain tau isoform and develop neuronal accumulations of tau in older animals. Examination of the old tau Tg mice showed that neuronal cells affected by tau accumulation induce co-accumulation of pT668-ACD. We speculate that in AD brains, extracellular Aβ deposition is accompanied by intracellular accumulation of pT668-ACD, followed by tau accumulation in the SP with dystrophic neurites and that neuronal cells affected by tau accumulation induce co-accumulation of pT668-ACD in NFTs. Thus, pT668-ACD is likely to mediate pathological interaction between Aβ and tau.     

Mice devoid of Tau have increased susceptibility to neuronal damage in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis

The abundant axonal microtubule-associated protein tau regulates microtubule and actin dynamics, thereby contributing to normal neuronal function. We examined whether mice deficient in tau (Tau(-/-)) or with high levels of human tau differ from wild-type (WT) mice in their susceptibility to neuroaxonal injury in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. After sensitization with MOG35-55, there was no difference in clinical disease course between human tau and WT mice, but Tau mice had more severe clinical disease and significantly more axonal damage in spinal cord white matter than those in WT mice. Axonal damage in gray matter correlated with clinical severity in individual mice. By immunoblot analysis, the early microtubule-associated protein-1b was increased 2-fold in the spinal cords of Tau mice with chronic experimental autoimmune encephalomyelitis versus naive Tau mice. This difference was not detected in comparable WT animals, which suggests that there was compensation for the loss of tau in the deficient mice. In addition, levels of the growth arrest-specific protein 7b, a tau-binding protein that is stabilized when bound to tau, were higher in WT than those in Tau(-/-) spinal cord samples. These data indicate that loss of tau exacerbates experimental autoimmune encephalomyelitis and suggest that maintaining tau integrity might reduce the axonal damage that occurs in inflammatory neurodegenerative diseases such as multiple sclerosis.     

Progressive tauopathy in P301S tau transgenic mice is associated with a functional deficit of the olfactory system

Multiple neurodegenerative disorders with tau pathology are characterised by the loss of memory and cognitive decline that can be associated with other symptoms including olfactory alterations that are often regarded as an early symptom of the diseases. Here, we have investigated whether olfactory dysfunction is present in the P301S human tau transgenic mice and if it is associated to tau pathology. Progressive tauopathy and neurodegeneration were noticeable in the olfactory bulb and piriform cortex at early age in the P301S human tau transgenic mice and olfactory sensitivity for social or non‐social odours was significantly impaired at 3 months of age, when the piriform cortex‐dependent odour‐cross habituation was also disrupted. The olfactory alterations in the P301S tau transgenic mouse line provide an in vivo system where to test the mechanism‐based therapies for the common and yet untreatable tauopathies.     

Transient cerebral ischemia induces site-specific hyperphosphorylation of tau protein

Neurofibrillary tangles (NFTs) are a pathological hallmark of many neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP17). However, the cellular origin and the consequence of the NFT formation are poorly understood. Epidemiological evidence suggests a much higher occurrence of dementia in stroke patients. This may represent the pathogenesis of sporadic AD, which accounts for the majority of AD occurrence. Here we show that after a transient cerebral ischemia, hyperphosphorylated tau accumulates in cortical neurons in a site-specific manner. The hyperphosphorylated tau presents a conformation similar to those present in human tauopathies, and colocalizes largely with signs of apoptosis. Our current study suggests that tau hyperphosphorylation may contribute to the brain damage induced by transient cerebral ischemia, and may be involved in the pathogenesis of neurodegenerative disorders in patients after stroke. Further, these results indicate that ischemic neuronal damage and apoptosis associates with tau hyperphosphorylation, and potentially NFTs formation. Finally, our results also suggest that neuronal apoptosis may be a therapeutic target in preventing tauopathy-related neurodegenerative diseases.     

Tau alternative splicing and frontotemporal dementia

A number of neurodegenerative diseases are characterized by the presence of abundant deposits containing Tau protein. Expression of the human tau gene is under complex regulation. Mutations in the tau gene have been identified in patients with frontotemporal lobe dementia. These mutations affect either biochemical/biophysical properties or the delicate balance of different splicing isoforms. In this review, we summarize recent advances in our understanding of genetics and molecular pathogenesis of tauopathies with the focus on frontotemporal lobe dementia. We review published studies on tau pre-mRNA splicing regulation. Understanding molecular mechanisms of tauopathies may help in developing effective therapies for neurodegenerative tauopathies and related disorders, including Alzheimer disease.     

Divergent phosphorylation pattern of tau in P301L tau transgenic mice

Aggregates of hyperphosphorylated tau are prominent in brains of patients with Alzheimer's disease or frontotemporal dementia (FTD). They have been reproduced in animal models following the identification of tau mutations in familial cases of FTD. This includes our previously generated transgenic model, pR5, which expresses FTD (P301L) mutant tau in neurons. The mice are characterized by tau aggregation including tangle (NFT) formation, memory impairment and mitochondrial dysfunction. In 8-month-old mice, S422 phosphorylation of tau is linked to NFT formation, however, a detailed analysis of tau solubility, phosphorylation and aggregation has not been done nor have the mice been monitored until a high age. Here, we undertook an analysis by immunohistochemistry, Gallyas impregnation and Western blotting of brains from 3 month- up to 20 month-old mice. NFTs first appeared at 6 months in the amygdala, followed by the CA1 region of the hippocampus. As the mice get older, the solubility of tau is decreased as determined by sequential extractions. Histological analysis revealed increased phosphorylation at the AT180, AT270 and 12E8 epitopes with ageing. The numbers of AT8-positive neurons increased from 3 to 6 months old. However, whereas S422 appeared only late and concomitantly with NFT formation, the only neurons left with AT8-reactivity at 20 months were those that had undergone NFT formation. As hyperphosphorylated tau continued to accumulate, the lack of AT8-reactivity suggests regulatory mechanisms in specifically dephosphorylating the AT8 epitope in the remaining neurons. Thus, differential regulation of phosphorylation is important for NFT formation in neurodegenerative diseases with tau pathology.