Phosphorylated PP2A (tyrosine 307) is associated with Alzheimer neurofibrillary pathology

Down-regulation of protein phosphatase 2A (PP2A) is thought to play a critical role in tau hyperphosphorylation in Alzheimer's disease (AD). In vitro phosphorylation of PP2A catalytic subunit at Y307 efficiently inactivates PP2A. A specific antibody against phosphorylated (p) PP2A (Y307) (PP2Ac-Yp307) was used to investigate possible PP2A down-regulation by known pathophysiological changes associated with AD, such as Abeta accumulation and oestrogen deficiency. Immunohistochemistry and immunofluorescence confocal microscopy showed an aberrant accumulation of PP2Ac-Yp307 in neurons that bear pretangles or tangles in the susceptible brain regions, such as the entorhinal cortical cortex and the hippocampus. Experimentally, increased PP2Ac-Yp307 was observed in mouse N2a neuroblastoma cells that stably express the human amyloid precursor protein with Swedish mutation (APPswe) compared with wild-type, and in the brains of transgenic APPswe/ presenilin (PS1, A246E) mice, which corresponded to the increased tau phosphorylation. Treating N2a cells with Abeta25-35 mimicked the changes of PP2Ac-Yp307 and tau phosphorylation in N2a APPswe cells. Knockout of oestrogen receptor (ER) alpha or ERbeta gave similar changes of PP2Ac-Yp307 level and tau phosphorylation in the mouse brain. Taken together, these findings suggest that increased PP2A phosphorylation (Y307) can be mediated by Abeta deposition or oestrogen deficiency in the AD brain, and consequently compromise dephosphorylation of abnormally hyperphosphorylated tau, and lead to neurofibrillary tangle formation.     

Sub-lethal levels of amyloid β-peptide oligomers decrease non-transferrin-bound iron uptake and do not potentiate iron toxicity in primary hippocampal neurons

Two major lesions are pathological hallmarks in Alzheimer's disease (AD): the presence of neurofibrillary tangles formed by intracellular aggregates of the hyperphosphorylated form of the cytoskeletal tau protein, and of senile plaques composed of extracellular aggregates of amyloid beta (Aβ) peptide. Current hypotheses regard soluble amyloid beta oligomers (AβOs) as pathological causative agents in AD. These aggregates cause significant calcium deregulation and mediate neurotoxicity by disrupting synaptic activity. Additionally, the presence of high concentrations of metal ions such as copper, zinc, aluminum and iron in neurofibrillary tangles and senile plaques, plus the fact that they accelerate the rate of formation of Aβ fibrils and AβOs in vitro, suggests that accumulation of these metals in the brain is relevant to AD pathology. A common cellular response to AβOs and transition metals such as copper and iron is the generation of oxidative stress, with the ensuing damage to cellular components. Using hippocampal neurons in primary culture, we report here the effects of treatment with AβOs on the (+)IRE and (-)IRE mRNA levels of the divalent metal transporter DMT1. We found that non-lethal AβOs concentrations decreased DMT1 (-)IRE without affecting DMT1 (+)IRE mRNA levels, and inhibited non-transferrin bound iron uptake. In addition, since both iron and AβOs induce oxidative damage, we studied whether their neurotoxic effects are synergistic. In the range of concentrations and times used in this study, AβOs did not potentiate iron-induced cell death while iron chelation did not decrease AβOs-induced cell death. The lack of synergism between iron and AβOs suggests that these two neurotoxic agents converge in a common target, which initiates signaling processes that promote neurodegeneration.     

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.     

Alzheimer neurofibrillary degeneration: significance, etiopathogenesis, therapeutics and prevention

Alzheimer disease (AD) is multi-factorial and heterogeneous. Independent of the aetiology, this disease is characterized clinically by chronic and progressive dementia and histopathologically by neurofibrillary degeneration of abnormally hyperphosphorylated tau seen as intraneuronal neurofibrillary tangles, neuropil threads and dystrophic neurites, and by neuritic (senile) plaques of beta-amyloid. The neurofibrillary degeneration is apparently required for the clinical expression of AD, and in related tauopathies it leads to dementia in the absence of amyloid plaques. While normal tau promotes assembly and stabilizes microtubules, the abnormally hyperphosphorylated tau sequesters normal tau, MAP1 and MAP2, and disrupts microtubules. The abnormal hyperphosphorylation of tau also promotes its self-assembly into tangles of paired helical and or straight filaments. Tau is phosphorylated by a number of protein kinases. Glycogen synthase kinase-3 (GSK-3) and cyclin dependent protein kinase 5 (cdk5) are among the kinases most implicated in the abnormal hyperphosphorylation of tau. Among the phosphatases which regulate the phosphorylation of tau, protein phosphatase-2A (PP-2A), the activity of which is down-regulated in AD brain, is by far the major enzyme. The inhibition of abnormal hyperphosphorylation of tau is one of the most promising therapeutic targets for the development of disease modifying drugs. A great advantage of inhibiting neurofibrillary degeneration is that it can be monitored by evaluating the levels of total tau and tau phosphorylated at various known abnormally hyperphosphorylated sites in the cerebrospinal fluid of patients, obtained by lumbar puncture. There are at least five subgroups of AD, each is probably caused by a different etiopathogenic mechanism. The AD subgroup identification of patients can help increase the success of clinical trials and the development of specific and potent disease modifying drugs.     

Changes in the detergent‐insoluble brain proteome linked to amyloid and tau in Alzheimer's Disease progression

Despite a key role of amyloid‐beta (Aβ) in Alzheimer's disease (AD), mechanisms that link Aβ plaques to tau neurofibrillary tangles and cognitive decline still remain poorly understood. The purpose of this study was to quantify proteins in the sarkosyl‐insoluble brain proteome correlated with Aβ and tau insolubility in the asymptomatic phase of AD (AsymAD) and through mild cognitive impairment (MCI) and symptomatic AD. Employing label‐free mass spectrometry‐based proteomics, we quantified 2711 sarkosyl‐insoluble proteins across the prefrontal cortex from 35 individual cases representing control, AsymAD, MCI and AD. Significant enrichment of Aβ and tau in AD was observed, which correlated with neuropathological measurements of plaque and tau tangle density, respectively. Pairwise correlation coefficients were also determined for all quantified proteins to Aβ and tau, across the 35 cases. Notably, six of the ten most correlated proteins to Aβ were U1 small nuclear ribonucleoproteins (U1 snRNPs). Three of these U1 snRNPs (U1A, SmD and U1‐70K) also correlated with tau consistent with their association with tangle pathology in AD. Thus, proteins that cross‐correlate with both Aβ and tau, including specific U1 snRNPs, may have potential mechanistic roles in linking Aβ plaques to tau tangle pathology during AD progression.     

Levels of mTOR and its downstream targets 4E-BP1, eEF2, and eEF2 kinase in relationships with tau in Alzheimer's disease brain

The pathogenesis of formation of neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) brains is unknown. One of the possibilities might be that translation of tau mRNA is aberrantly regulated in AD brains. In the current study, levels of various translation control elements including total and phosphorylated (p) forms of mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4E binding protein 1 (4E-BP1), eukaryotic elongation factor 2 (eEF2), and eEF2 kinase were investigated in relationship with tau in homogenates of the medial temporal cortex from 20 AD and 10 control brains. We found that levels of p-mTOR (Ser2481), and p-4E-BP1 (Thr70 and Ser65) dramatically increase in AD, and are positively significantly correlated with total tau and p-tau. Levels of p-eEF2K were significantly increased, and total eEF2 significantly decreased in AD, when compared to controls. The changes of p-mTOR (2481), p-4E-BP1, and p-eEF2 were immunohistochemically confirmed to be in neurons of AD brains. This suggested that there are obvious abnormalities of elements related with translation control in AD brain and their aberrant changes may up-regulate the translation of tau mRNA, contributing to hyperphosphorylated tau accumulation in NFT-bearing neurons.     

MARKing tau for tangles and toxicity

In healthy neurons, tau proteins regulate microtubule function in the axon. In the brains of individuals with Alzheimer's disease, tau is hyperphosphorylated and aggregated into intraneuronal deposits called neurofibrillary tangles (NFTs). Hyperphosporylation dislodges tau from the microtubule surface, potentially resulting in compromised axonal integrity and the accumulation of toxic tau peptides. Recent biochemical and animal model studies have re-evaluated tau phosphorylation and other aspects of neurofibrillar pathology. The results indicate that phosphorylation of tau's microtubule-binding domain by the protein kinase MARK primes tau for hyperphosphorylation by the kinases GSK-3 and Cdk5, which in turn triggers the aggregation of tau into filaments and tangles. Toxic consequences for the neuron might be exacerbated by tangle formation but are already evident during the early steps of the process.     

Role of cdk5 in the pathogenesis of Alzheimer's disease

Alzheimer's disease (AD) is characterized by two pathological hallmarks, namely, senile plaques and neurofibrillary tangles (NFTs). The former are mainly composed of amyloid-beta peptides (Abeta) while the latter consists mainly of filaments of hyperphosphorylated tau. Cyclin-dependent kinase 5 (cdk5) has been implicated not only in the tangle pathology, but recent data also implicate cdk5 in the generation of Abeta peptides. Since both Abeta peptides and NFTs are believed to play a role in neurodegeneration in AD, this proline-directed serine/threonine protein kinase is likely to contribute to the pathogenesis of AD. In vitro and in vivo animal data demonstrate the ability of cdk5 to induce phosphorylation and aggregation of tau, and NFT deposition and neurodegeneration. Findings from AD brain samples also show an elevated cdk5 activity and conditions that support the activation of cdk5. Evidence for the role of cdk5 in regulating Abeta production is just emerging. The mechanisms for this potentially damaging activity of cdk5 are largely unknown although amyloid precursor protein and presenilin-1 are both cdk5 substrates.     

Tau PET Imaging for Staging of Alzheimer's Disease in Down Syndrome

Alzheimer's disease (AD) pathology and early‐onset dementia develop almost universally in Down syndrome (DS). AD is defined neuropathologically by the presence of extracellular plaques of aggregated amyloid β protein and intracellular neurofibrillary tangles (NFTs) of aggregated hyperphosphorylated tau protein. The development of radiolabeled positron emission tomography (PET) ligands for amyloid plaques and tau tangles enables the longitudinal assessment of the spatial pattern of their accumulation in relation to symptomatology. Recent work indicates that amyloid pathology develops 15–20 years before neurodegeneration and symptom onset in the sporadic and autosomal dominant forms of AD, while tau pathology correlates more closely with symptomatic stages evidenced by cognitive decline and dementia. Recent work on AD biomarkers in DS illustrates similarities between DS and sporadic AD. It may soon be possible to apply recently developed staging classifications to DS to obtain a more nuanced understanding of the development AD in DS and to provide more accurate diagnosis and prognosis in the clinic.     

Acetylcholine receptors and tau phosphorylation

Alzheimer's disease (AD) is characterized by the presence, in the brain of the patients, of two aberrant structures: intracellular neurofibrillary tangles (NFTs), containing an abnormal hyperphosphorylated form of tau protein, and extracellular senile plaques (SPs), mainly composed by fibrillar amyloid beta peptide. Another feature of AD is the neurodegeneration and dysfunction of basal forebrain cholinergic system. A possible connection among those AD characteristics could occur. Thus, the purpose of this short review is to summarize the involvement of nicotinic (nAChR) and muscarinic (mAChR) receptors on tau phosphorylation, in a direct way, or through the previous interaction of some of these receptors with amyloid beta. Several studies have demonstrated that nAChR activation results in a significantly increase of tau phosphorylation, whereas mAChR activation, may prevent tau phosphorylation.     

Copper in the brain and Alzheimer’s disease

Alzheimer’s disease (AD) is the most common form of neurodegenerative disease. The brain is particularly vulnerable to oxidative damage induced by unregulated redox-active metals such as copper and iron, and the brains of AD patients display evidence of metal dyshomeostasis and increased oxidative stress. The colocalisation of copper and amyloid β (Aβ) in the glutamatergic synapse during NMDA-receptor-mediated neurotransmission provides a microenvironment favouring the abnormal interaction of redox-potent Aβ with copper under conditions of copper dysregulation thought to prevail in the AD brain, resulting in the formation of neurotoxic soluble Aβ oligomers. Interactions between Aβ oligomers and copper can further promote the aggregation of Aβ, which is the core component of extracellular amyloid plaques, a central pathological hallmark of AD. Copper dysregulation is also implicated in the hyperphosphorylation and aggregation of tau, the main component of neurofibrillary tangles, which is also a defining pathological hallmark of AD. Therefore, tight regulation of neuronal copper homeostasis is essential to the integrity of normal brain functions. Therapeutic strategies targeting interactions between Aβ, tau and metals to restore copper and metal homeostasis are discussed.     

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.     

Uncharged thioflavin-T derivatives bind to amyloid-beta protein with high affinity and readily enter the brain

In vivo assessment of the beta-sheet proteins deposited in amyloid plaques (A beta peptide) or neurofibrillary tangles (tau protein) presents a target for the development of biological markers for Alzheimer's disease (AD). In an effort to develop in vivo beta-sheet imaging probes, derivatives of thioflavin-T (ThT) were synthesized and evaluated. These compounds lack the positively charged quaternary heterocyclic nitrogen of ThT and are therefore uncharged at physiological pH. They are 600-fold more lipophilic than ThT. These ThT derivatives bind to A beta(1-40) fibrils with higher affinity (Ki = 20.2 nM) than ThT (Ki = 890 nM). The uncharged ThT derivatives stained both plaques and neurofibrillary tangles in post-mortem AD brain, showing some preference for plaque staining. A carbon-11 labeled compound, [N-methyl-11C]6-Me-BTA-1, was prepared, and its brain entry and clearance were studied in Swiss-Webster mice. This compound entered the brain at levels comparable to commonly used neuroreceptor imaging agents (0.223 %ID-kg/g or 7.61 %ID/g at 2 min post-injection) and showed good clearance of free and non-specifically bound radioactivity in normal rodent brain tissue (brain clearance t(1,2) = 20 min). The combination of relatively high affinity for amyloid, specificity for staining plaques and neurofibrillary tangles in post-mortem AD brain, and good brain entry and clearance makes [N-methyl-11C]6-Me-BTA-1 a promising candidate as an in vivo positron emission tomography (PET) beta-sheet imaging agent.     

N epsilon-carboxymethyllysine in brain aging, diabetes mellitus, and Alzheimer's disease

Oxidative stress has been implicated in the pathophysiology of Alzheimer's disease (AD) and diabetes mellitus (DM). N epsilon-carboxymethyllysine (CML) is an advanced glycation end product (AGE) recently found to be associated with oxidative stress mechanisms. Using immunocytochemical methods we examined the distribution of CML in brain tissue from AD and DM subjects and aging controls. CML reactivity was present in the cytoplasm of neurons, but there were marked differences in the intensity of expression, number of cells, and topographical distribution. CML expression was higher in hippocampus than in frontal and temporal cortex. In the hippocampus, neuronal and, to an extent, glial expression was more marked in CA3 and CA4 than in CA1 and CA2. In AD, CML was found to be coexpressed with tau protein, showing the similar neurofibrillary tangle shape, as well as in neuritic plaques but not in the core of amyloid plaques. We noted an increasing degree of CML expression such that the highest reactivity was evident in those with both AD and DM, followed by AD, DM, and aging controls. There was an inverse relationship between Braak staging and topography of CML expression. Although DM cases did not show Abeta deposition or neurofibrillary tangles, these findings suggest increased CML expression is not limited to AD. Nonetheless, high CML expression in AD with coexistent DM suggests there are additive effects compared with AD alone. It is plausible that the microangiopathy also associated with DM could worsen AD pathogenesis.     

PKR knockout in the 5xFAD model of Alzheimer's disease reveals beneficial effects on spatial memory and brain lesions

Brain lesions in Alzheimer's disease (AD) include amyloid plaques made of Aβ peptides and neurofibrillary tangles composed of hyperphosphorylated tau protein with synaptic and neuronal loss and neuroinflammation. Aβ oligomers can trigger tau phosphorylation and neuronal alterations through activation of neuronal kinases leading to progressive cognitive decline. PKR is a ubiquitous pro‐apoptotic serine/threonine kinase, and levels of activated PKR are increased in AD brains and AD CSF. In addition, PKR regulates negatively memory formation in mice. To assess the role of PKR in an AD in vivo model, we crossed 5xFAD transgenic mice with PKR knockout (PKRKO) mice and we explored the contribution of PKR on cognition and brain lesions in the 5xFAD mouse model of AD as well as in neuron–microglia co‐cultures exposed to the innate immunity activator lipopolysaccharide (LPS). Nine‐month‐old double‐mutant mice revealed significantly improved memory consolidation with the new object location test, starmaze test, and elevated plus maze test as compared to 5xFAD mice. Brain amyloid accumulation and BACE1 levels were statistically decreased in double‐mutant mice. Apoptosis, neurodegeneration markers, and synaptic alterations were significantly reduced in double‐mutant mice as well as neuroinflammation markers such as microglial load and brain cytokine levels. Using cocultures, we found that PKR in neurons was essential for LPS microglia‐induced neuronal death. Our results demonstrate the clear involvement of PKR in abnormal spatial memory and brain lesions in the 5xFAD model and underline its interest as a target for neuroprotection in AD.     

Monitoring protein aggregation and toxicity in Alzheimer's disease mouse models using in vivo imaging

Aggregation of amyloid beta peptide into senile plaques and hyperphosphorylated tau protein into neurofibrillary tangles in the brain are the pathological hallmarks of Alzheimer's disease. Despite over a century of research into these lesions, the exact relationship between pathology and neurotoxicity has yet to be fully elucidated. In order to study the formation of plaques and tangles and their effects on the brain, we have applied multiphoton in vivo imaging of transgenic mouse models of Alzheimer's disease. This technique allows longitudinal imaging of pathological aggregation of proteins and the subsequent changes in surrounding neuropil neurodegeneration and recovery after therapeutic interventions.     

Alzheimer-specific epitopes of tau represent lipid peroxidation-induced conformations

Several recent studies support a link between tau protein phosphorylation and adduction of tau by reactive carbonyls. Indeed, the phosphorylation-dependent adduction of tau by carbonyl products resulting from lipid peroxidation creates the neurofibrillary tangle-related antigen, Alz50. To determine whether epitopes of carbonyl-modified tau are major conformational changes associated with neurofibrillary tangle formation, we examined seven distinct antibodies raised against neurofibrillary tangles that recognize unique epitopes of tau in Alzheimer disease. Consistently, all seven antibodies recognize tau more strongly (4- to 34-fold) after treatment of normal tau with the reactive carbonyl, 4-hydroxy-2-nonenal (HNE), but only when tau is in the phosphorylated state. These findings not only support the idea that oxidative stress is involved in neurofibrillary tangle formation occurring in brains of Alzheimer disease patients, but also show, for the first time, that HNE modifications of tau promote and contribute to the generation of the major conformational properties defining neurofibrillary tangles.