In vivo evidence of CHIP up-regulation attenuating tau aggregation

The carboxyl terminus of heat-shock cognate (Hsc)70-interacting protein (CHIP) is a ubiquitin E3 ligase that can collaborate with molecular chaperones to facilitate protein folding and prevent protein aggregation. Previous studies showed that, together with heat-shock protein (Hsp)70, CHIP can regulate tau ubiquitination and degradation in a cell culture system. Ubiquitinated tau is one component in neurofibrillary tangles (NFTs), which are a major histopathological feature of Alzheimer's disease (AD). However, the precise sequence of events leading to NFT formation and the mechanisms involved remain unclear. To confirm CHIP's role in suppressing NFT formation in vivo, we performed a quantitative analysis of CHIP in human and mouse brains. We found increased levels of CHIP and Hsp70 in AD compared with normal controls. CHIP levels in both AD and controls corresponded directly to Hsp90 levels, but not to Hsp70 or Hsc70 levels. In AD samples, CHIP was inversely proportional to sarkosyl-insoluble tau accumulation. In a JNPL3 mouse brain tauopathy model, CHIP was widely distributed but weakly expressed in spinal cord, which was the most prominent region for tau inclusions and neuronal loss. Protein levels of CHIP in cerebellar regions of JNPL3 mice were significantly higher than in non-transgenic littermates. Human tau was more highly expressed in this region of mouse brains, but only moderate levels of sarkosyl-insoluble tau were detected. This was confirmed when increased insoluble tau accumulation was found in mice lacking CHIP. These findings suggest that increases in CHIP may protect against NFT formation in the early stages of AD. If confirmed, this would indicate that the quality-control machinery in a neuron might play an important role in retarding the pathogenesis of tauopathies.     

Transgenic mice expressing mutant (N279K) human tau show mutation dependent cognitive deficits without neurofibrillary tangle formation

Mutations in the tau gene, which is located on chromosome 17, were found causative for autosomal dominantly inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). To determine if cognitive deficits could be caused by tau mutations, two transgenic mouse lines were generated expressing a four-repeat isoform of human tau or its mutant, containing one of the FTDP-17 mutations (WILD mice and N279K mice). In open field test, N279K mice showed hyperactivity in locomotion and rearing. In prepulse inhibition test, N279K mice but not Wild mice showed significant deficits. Both transgenic mice, especially N279K mice, showed impairment in acquisition of spatial learning in Morris water maze. Although both N279K mice and Wild mice acquired passive avoidance as well as non-transgenic mice, N279K mice but not Wild mice showed severe deficits in acquisition of active avoidance. Histological analysis of the present mutant mice did not show any signs of neurofibrillary tangle formations in the brain, and cognitive dysfunction seemed to precede such neuropathological changes or occur independently from them. The behavioral phenotype of N279K mice mimics features of human FTDP-17 and provides a basic model for elucidating mechanisms underlying cognitive deficits in not only FTDP-17, but also diverse tauopathies.     

Passive Immunization with Phospho-Tau Antibodies Reduces Tau Pathology and Functional Deficits in Two Distinct Mouse Tauopathy Models

In Alzheimer’s disease (AD), an extensive accumulation of extracellular amyloid plaques and intraneuronal tau tangles, along with neuronal loss, is evident in distinct brain regions. Staging of tau pathology by postmortem analysis of AD subjects suggests a sequence of initiation and subsequent spread of neurofibrillary tau tangles along defined brain anatomical pathways. Further, the severity of cognitive deficits correlates with the degree and extent of tau pathology. In this study, we demonstrate that phospho-tau (p-tau) antibodies, PHF6 and PHF13, can prevent the induction of tau pathology in primary neuron cultures. The impact of passive immunotherapy on the formation and spread of tau pathology, as well as functional deficits, was subsequently evaluated with these antibodies in two distinct transgenic mouse tauopathy models. The rTg4510 transgenic mouse is characterized by inducible over-expression of P301L mutant tau, and exhibits robust age-dependent brain tau pathology. Systemic treatment with PHF6 and PHF13 from 3 to 6 months of age led to a significant decline in brain and CSF p-tau levels. In a second model, injection of preformed tau fibrils (PFFs) comprised of recombinant tau protein encompassing the microtubule-repeat domains into the cortex and hippocampus of young P301S mutant tau over-expressing mice (PS19) led to robust tau pathology on the ipsilateral side with evidence of spread to distant sites, including the contralateral hippocampus and bilateral entorhinal cortex 4 weeks post-injection. Systemic treatment with PHF13 led to a significant decline in the spread of tau pathology in this model. The reduction in tau species after p-tau antibody treatment was associated with an improvement in novel-object recognition memory test in both models. These studies provide evidence supporting the use of tau immunotherapy as a potential treatment option for AD and other tauopathies.     

Brain levels of CDK5 activator p25 are not increased in Alzheimer's or other neurodegenerative diseases with neurofibrillary tangles

Elevated levels of p25 and constitutive activation of CDK5 have been observed in AD brains. This has led to the hypothesis that increased p25 levels could promote neurofibrillary tangles (NFT) through CDK5-mediated hyperphosphorylation of tau, the principal component of NFTs. We examined p25 immunoreactivity in brains from sporadic and familial AD cases, as well as other neurologic diseases that exhibit NFT, such as Down's syndrome (DS), Pick's disease (Pick), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD). Neither the p25 immunoreactivity nor the p25/p35 ratio was elevated in the AD brains or in the other tauopathies (n = 34) compared with controls (n = 11). Although Abeta peptides have been suggested to activate calpain-mediated cleavage of p35 to p25 in cultured neurons, p25 levels in brains of TgCRND8 mice, which express high levels of brain Abeta peptides, were similar to those of non-Tg littermates. Our data suggest that high Abeta levels in brain do not activate p35 proteolysis, and p25 is unlikely to be a causative agent for NFT formation in AD or other tauopathies.     

Tau Proteins and Tauopathies in Alzheimer’s Disease

Alzheimer’s disease (AD) is characterized by progressive memory loss and cognitive function deficits. There are two major pathological hallmarks that contribute to the pathogenesis of AD which are the presence of extracellular amyloid plaques composed of amyloid-β (Aβ) and intracellular neurofibrillary tangles composed of hyperphosphorylated tau. Despite extensive research that has been done on Aβ in the last two decades, therapies targeting Aβ were not very fruitful at treating AD as the efficacy of Aβ therapies observed in animal models is not reflected in human clinical trials. Hence, tau-directed therapies have received tremendous attention as the potential treatments for AD. Tauopathies are closely correlated with dementia and immunotherapy has been effective at reducing tau pathology and improving cognitive deficits in animal models. Thus, in this review article, we discussed the pathological mechanism of tau proteins, the key factors contributing to tauopathies, and therapeutic approaches for tauopathies in AD based on the recent progress in tau-based research.     

Tau deficiency induces parkinsonism with dementia by impairing APP-mediated iron export

The microtubule-associated protein tau has risk alleles for both Alzheimer's disease and Parkinson's disease and mutations that cause brain degenerative diseases termed tauopathies. Aggregated tau forms neurofibrillary tangles in these pathologies, but little is certain about the function of tau or its mode of involvement in pathogenesis. Neuronal iron accumulation has been observed pathologically in the cortex in Alzheimer's disease, the substantia nigra (SN) in Parkinson's disease and various brain regions in the tauopathies. Here we report that tau-knockout mice develop age-dependent brain atrophy, iron accumulation and SN neuronal loss, with concomitant cognitive deficits and parkinsonism. These changes are prevented by oral treatment with a moderate iron chelator, clioquinol. Amyloid precursor protein (APP) ferroxidase activity couples with surface ferroportin to export iron, but its activity is inhibited in Alzheimer's disease, thereby causing neuronal iron accumulation. In primary neuronal culture, we found loss of tau also causes iron retention, by decreasing surface trafficking of APP. Soluble tau levels fall in affected brain regions in Alzheimer's disease and tauopathies, and we found a similar decrease of soluble tau in the SN in both Parkinson's disease and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model. These data suggest that the loss of soluble tau could contribute to toxic neuronal iron accumulation in Alzheimer's disease, Parkinson's disease and tauopathies, and that it can be rescued pharmacologically.     

Shift in the ratio of three-repeat tau and four-repeat tau mRNAs in individual cholinergic basal forebrain neurons in mild cognitive impairment and Alzheimer's disease

Molecular mechanisms underlying tauopathy remain undetermined. In the current study, single cell gene expression profiling was coupled with custom-designed cDNA array analysis to evaluate tau expression and other cytoskeletal elements within individual neuronal populations in patients with no cognitive impairment (NCI), mild cognitive impairment (MCI), and Alzheimer's disease (AD). Results revealed a shift in the ratio of three-repeat tau (3Rtau) to four-repeat tau (4Rtau) mRNAs within individual human cholinergic basal forebrain (CBF) neurons within nucleus basalis (NB) and CA1 hippocampal neurons during the progression of AD, but not during normal aging. A shift in 3Rtau to 4Rtau may precipitate a cascade of events in the selective vulnerability of neurons, ultimately leading to frank neurofibrillary tangle (NFT) formation in tauopathies including 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.     

mTOR regulates tau phosphorylation and degradation: implications for Alzheimer's disease and other tauopathies

Accumulation of tau is a critical event in several neurodegenerative disorders, collectively known as tauopathies, which include Alzheimer's disease and frontotemporal dementia. Pathological tau is hyperphosphorylated and aggregates to form neurofibrillary tangles. The molecular mechanisms leading to tau accumulation remain unclear and more needs to be done to elucidate them. Age is a major risk factor for all tauopathies, suggesting that molecular changes contributing to the aging process may facilitate tau accumulation and represent common mechanisms across different tauopathies. Here, we use multiple animal models and complementary genetic and pharmacological approaches to show that the mammalian target of rapamycin (mTOR) regulates tau phosphorylation and degradation. Specifically, we show that genetically increasing mTOR activity elevates endogenous mouse tau levels and phosphorylation. Complementary to it, we further demonstrate that pharmacologically reducing mTOR signaling with rapamycin ameliorates tau pathology and the associated behavioral deficits in a mouse model overexpressing mutant human tau. Mechanistically, we provide compelling evidence that the association between mTOR and tau is linked to GSK3β and autophagy function. In summary, we show that increasing mTOR signaling facilitates tau pathology, while reducing mTOR signaling ameliorates tau pathology. Given the overwhelming evidence that reducing mTOR signaling increases lifespan and healthspan, the data presented here have profound clinical implications for aging and tauopathies and provide the molecular basis for how aging may contribute to tau pathology. Additionally, these results provide preclinical data indicating that reducing mTOR signaling may be a valid therapeutic approach for tauopathies.     

Glycosaminoglycans and beta-amyloid,prion and tau peptides in neurodegenerative diseases

Protein aggregation into dense filamentous inclusions is a characteristic feature of many etiologically diverse neurodegenerative disorders including Alzheimer's disease (AD), spongiform encephalopathies, and tauopathies. Thus, beta-amyloid peptide (Abeta) accumulates within senile amyloid plaques in AD, protease-resistant prion protein constitutes the amyloid deposits in spongiform encephalopathies and tau protein gives rise to neurofibrillary tangles (NFT) both in AD and in tauopathies. Curiously, these abnormal protein inclusions contain, in addition to their major peptide components, some associated sulfated glycosaminoglycans (sGAG). Here we discuss the proposal that the binding of sGAG to aggregate-forming peptides may modify the pathogenic process depending on their subcellular localization.     

Calpain-mediated tau cleavage: a mechanism leading to neurodegeneration shared by multiple tauopathies

Tau dysfunction has been associated with a host of neurodegenerative diseases called tauopathies. These diseases share, as a common pathological hallmark, the presence of intracellular aggregates of hyperphosphorylated tau in affected brain areas. Aside from tau hyperphosphorylation, little is known about the role of other posttranslational modifications in tauopathies. Recently, we obtained data suggesting that calpain-mediated tau cleavage leading to the generation of a neurotoxic tau fragment might play an important role in Alzheimer's disease. In the current study, we assessed the presence of this tau fragment in several tauopathies. Our results show high levels of the 17-kDa tau fragment and enhanced calpain activity in the temporal cortex of AD patients and in brain samples obtained from patients with other tauopathies. In addition, our data suggest that this fragment could partially inhibit tau aggregation. Conversely, tau aggregation might prevent calpain-mediated cleavage, establishing a feedback circuit that might lead to the accumulation of this toxic tau fragment. Collectively, these data suggest that the mechanism underlying the generation of the 17-kDa neurotoxic tau fragment might be part of a conserved pathologic process shared by multiple tauopathies.     

Fibroblast growth factor 10 ameliorates neurodegeneration in mouse and cellular models of Alzheimer's disease via reducing tau hyperphosphorylation and neuronal apoptosis

Alzheimer's disease (AD) is characterized with senile plaques formed by Aβ deposition, and neurofibrillary tangles composed of hyperphosphorylated tau protein, which ultimately lead to cognitive impairment. Despite the heavy economic and life burdens faced by the patients with AD, effective treatments are still lacking. Previous studies have reported the neuroprotective effects of FGF10 in CNS diseases, but its role in AD remains unclear. In this study, we demonstrated that FGF10 levels were reduced in the serum of AD patients, as well as in the brains of 3xTg-AD mice and APPswe-transfected HT22 cells, suggesting a close relationship between FGF10 and AD. Further investigations revealed that intranasal delivery of FGF10 improved cognitive functions in 3xTg-AD mice. Additionally, FGF10 treatment reduced tau hyperphosphorylation and neuronal apoptosis, thereby mitigating neuronal cell damage and synaptic deficits in the cortex and hippocampus of 3xTg-AD mice, as well as APPswe-transfected HT22 cells. Furthermore, we evaluated the therapeutic potential of FGF10 gene delivery for treating AD symptoms and pathologies. Tail vein delivery of the FGF10 gene using AAV9 improved cognitive and neuronal functions in 3xTg-AD mice. Similarly, endogenous FGF10 overexpression ameliorated tau hyperphosphorylation and neuronal apoptosis in the cortex and hippocampus of 3xTg-AD mice. Importantly, we confirmed that the FGFR2/PI3K/AKT signaling pathway was activated following intranasal FGF10 delivery and AAV9-mediated FGF10 gene delivery in 3xTg-AD mice and APPswe-transfected HT22 cells. Knockdown of FGFR2 attenuated the protective effect of FGF10. Collectively, these findings suggest that intranasal delivery of FGF10 and AAV9-mediated FGF10 gene delivery could be a promising disease-modifying therapy for AD.     

Cortical and Hippocampal Neurons from Truncated Tau Transgenic Rat Express Multiple Markers of Neurodegeneration

Transition of protein tau from physiologically unfolded to misfolded state represent enigmatic step in the pathogenesis of tauopathies including Alzheimer’s disease (AD). Major molecular events playing role in this process involve truncation and hyperphosphorylation of tau protein, which are accompanied by redox imbalance followed by functional deterioration of neuronal network. Recently we have developed transgenic rat model showing that expression of truncated tau causes neurofibrillary degeneration similar to that observed in brain of AD sufferers. Consequently we tested cortical and hippocampal neuronal cultures extracted from this model as a convenient tool for development of molecules able to target the mechanisms leading to and/or enhancing the process of neurodegeneration. Here we document three major pathological features typical for tauopathies and AD in cortical and hippocampal neurons from transgenic rat in vitro. First, an increased accumulation of human truncated tau in neurons; second, the hyperphosphorylation of truncated tau on the epitopes characteristic of AD (Ser202/Thr205 and Thr231); and third, increased vulnerability of the neurons to nitrative and oxidative stress. Our results show that primary neurons expressing human truncated tau could represent a cellular model for targeting tau related pathological events, namely, aberrant tau protein accumulation, tau hyperphosphorylation, and oxidative/nitrative damage. These characteristics make the model particularly suitable for detailed study of molecular mechanisms of tau induced neurodegeneration and easily applicable for drug screening.     

Tau蛋白高度磷酸化致阿尔茨海默病动物模型研究进展

阿尔茨海默病（Alzheimer＇sdisease,AD）是老年人中最常见的神经退行性疾病,以过度磷酸化tau蛋白为核心形成的神经原纤维缠结为AD的主要病理特征之一。近年来对tau蛋白磷酸化的研究备受关注。在AD的实验研究中,探索理想的AD动物模型对于明确AD的病因、发病机制及药物的研发等方面起关键作用。本文对Tau蛋白磷酸化致AD主要动物模型的研究进展进行了综述,包括Tau转基因动物模型、激酶和磷酸化酶系统失衡致Tau蛋白过度磷酸化损伤模型、降低Tau蛋白糖基化致Tau过度磷酸化模型等。     

New Age of Neuroproteomics in Alzheimer’s Disease Research

Alzheimer’s disease (AD) is the leading cause of dementia, a condition that gradually destroys brain cells and leads to progressive decline in mental functions. The disease is characterized by accumulation of misfolded neuronal proteins, amyloid and tau, into insoluble aggregates known as extracellular senile plaques and intracellular neurofibrillary tangles, respectively. However, only tau pathology appears to correlate with the progression of the disease and it is believed to play a central role in the progression of neurodegeneration. In AD, tau protein undergoes various types of posttranslational modifications, most notably hyperphosphorylation and truncation. Using four proteomics approaches we aimed to uncover the key steps leading to neurofibrillary degeneration and thus to identify therapeutic targets for AD. Functional neuroproteomics was employed to generate the first transgenic rat model of AD by expressing a truncated misordered form of tau, “Alzheimer’s tau”. The rat model showed that Alzheimer’s tau toxic gain of function is responsible for the induction of abnormal tau cascade and is the driving force in the development of neurofibrillary degeneration. Structural neuroproteomics allowed us to determine partial 3D structure of the Alzheimer’s filament core at a resolution of 1.6 Å. Signaling neuroproteomics data lead to the identification and characterization of relevant phosphosites (the tau phosphosignalome) contributing to neurodegeneration. Interaction neuroproteomics revealed links to a new group of proteins interacting with Alzheimer’s tau (tau interactome) under normal and pathological conditions, which would provide novel drug targets and novel biomarkers for treatment of AD and other tauopathies.     

Mouse models of Alzheimer's disease: a quest for plaques and tangles

Many genetically altered mice have been designed to help understand the role of specific gene mutations in the pathogenesis of Alzheimer's disease (AD) based on the realization that specific mutations in the genes for amyloid precursor protein--the presenilins and tau--are associated with early-onset familial AD or, in the case of tau mutations, other neurodegenerative diseases with neurofibrillary tangles. However, attempts to reproduce the neuropathology of AD in the mouse have been frustrating. Transgenic designs emphasizing amyloid precursor protein produced mice that develop amyloid plaques, but neurodegeneration and neurofibrillary tangles failed to form. Strategies emphasizing tau resulted in increased phosphorylation of tau and tangle formation, although amyloid plaques were absent. Nevertheless, crossing transgenic animals expressing mutated tau and amyloid precursor protein has produced a mouse that closely recapitulates the neuropathology of AD. A review of the various murine models, their role in understanding the pathogenesis of AD and their use in testing therapeutic regimens, is provided.     

Acetylation changes tau interactome to degrade tau in Alzheimer’s disease animal and organoid models

Alzheimer's disease (AD) is an age‐related neurodegenerative disease. The most common pathological hallmarks are amyloid plaques and neurofibrillary tangles in the brain. In the brains of patients with AD, pathological tau is abnormally accumulated causing neuronal loss, synaptic dysfunction, and cognitive decline. We found a histone deacetylase 6 (HDAC6) inhibitor, CKD‐504, changed the tau interactome dramatically to degrade pathological tau not only in AD animal model (ADLP^APT) brains containing both amyloid plaques and neurofibrillary tangles but also in AD patient‐derived brain organoids. Acetylated tau recruited chaperone proteins such as Hsp40, Hsp70, and Hsp110, and this complex bound to novel tau E3 ligases including UBE2O and RNF14. This complex degraded pathological tau through proteasomal pathway. We also identified the responsible acetylation sites on tau. These dramatic tau‐interactome changes may result in tau degradation, leading to the recovery of synaptic pathology and cognitive decline in the ADLP^APT mice.     

Pimozide reduces toxic forms of tau in TauC3 mice via 5′ adenosine monophosphate‐activated protein kinase‐mediated autophagy

In neurodegenerative diseases like Alzheimer's disease (AD), tau is hyperphosphorylated and forms aggregates and neurofibrillary tangles in affected neurons. Autophagy is critical to clear the aggregates of disease‐associated proteins and is often altered in patients and animal models of AD. Because mechanistic target of rapamycin (mTOR) negatively regulates autophagy and is hyperactive in the brains of patients with AD, mTOR is an attractive therapeutic target for AD. However, pharmacological strategies to increase autophagy by targeting mTOR inhibition cause various side effects. Therefore, autophagy activation mediated by non‐mTOR pathways is a new option for autophagy‐based AD therapy. Here, we report that pimozide activates autophagy to rescue tau pathology in an AD model. Pimozide increased autophagic flux through the activation of the AMPK‐Unc‐51 like autophagy activating kinase 1 (ULK1) axis, but not of mTOR, in neuronal cells, and this function was independent of dopamine D2 receptor inhibition. Pimozide reduced levels of abnormally phosphorylated tau aggregates in neuronal cells. Further, daily intraperitoneal (i.p.) treatment of pimozide led to a recovery from memory deficits of TauC3 mice expressing a caspase‐cleaved form of tau. In the brains of these mice, we found increased phosphorylation of AMPK1 and ULK1, and reduced levels of the soluble oligomers and NP40‐insoluble aggregates of abnormally phosphorylated tau. Together, these results suggest that pimozide rescues memory impairments in TauC3 mice and reduces tau aggregates by increasing autophagic flux through the mTOR‐independent AMPK‐ULK1 axis.     

Secretion of human tau fragments resembling CSF-tau in Alzheimer’s disease is modulated by the presence of the exon 2 insert

Abnormal tau cleavage is prominent in the neurofibrillary degeneration characteristic of Alzheimer’s disease (AD) and related tauopathies. We recently showed that cleaved human tau is secreted by specific mechanisms when overexpressed. Here we examined the effect of expressing N-terminal and full length tau constructs in transiently and stably transfected neuronal lines. We show that secreted tau exhibits a cleavage pattern similar to CSF-tau from human AD patients and that tau secretion is specifically inhibited by the presence of the exon 2 insert. These results suggest that tau secretion may play a hitherto unsuspected role in AD and related tauopathies.