Tangles In Alzheimer's Disease Brain Research Articles

Native PLGA nanoparticles attenuate Aβ-seed induced tau aggregation under in vitro conditions: potential implication in Alzheimer's disease pathology.

Evidence suggests that beta-amyloid (Aβ)-induced phosphorylation/aggregation of tau protein plays a critical role in the degeneration of neurons and development of Alzheimer's disease (AD), the most common cause of dementia affecting the elderly population. Many studies have pursued a variety of small molecules, including nanoparticles conjugated with drugs to interfere with Aβ and/or tau aggregation/toxicity as an effective strategy for AD treatment. We reported earlier that FDA approved PLGA nanoparticles without any drug can attenuate Aβ aggregation/toxicity in cellular/animal models of AD. In this study, we evaluated the effects of native PLGA on Aβ seed-induced aggregation of tau protein using a variety of biophysical, structural and spectroscopic approaches. Our results show that Aβ1-42 seeds enhanced aggregation of tau protein in the presence and absence of heparin and the effect was attenuated by native PLGA nanoparticles. Interestingly, PLGA inhibited aggregation of both 4R and 3R tau isoforms involved in the formation of neurofibrillary tangles in AD brains. Furthermore, Aβ seed-induced tau aggregation in the presence of arachidonic acid was suppressed by native PLGA. Collectively, our results suggest that native PLGA nanoparticles can inhibit the Aβ seed-induced aggregation of different tau protein isoforms highlighting their therapeutic implication in the treatment of AD.

Approaches to quantitative analysis of the PET tau radiotracer [18F]AV1451

Tau PET images in Alzheimer's disease (AD) demonstrate a pattern of localized, asymmetric cortical uptake roughly consistent with postmortem studies of the distribution of neurofibrillary tangles in AD brain. This heterogeneous abnormal uptake creates difficulties for volume of interest sampling which may result in a brain region which has distinct subregions with both abnormal and normal radiotracer uptake. The objective of this study is to explore strategies for optimal quantification of [18F]AV1451. [18F]AV1451 PET images from 10 subjects (3 AD, 5 PD, 2 CTE) were co-registered with subject's MRI and both images spatially normalized into standard space. PET uptake was extracted in different brain regions for grey matter only. Voxel-wise SUVr images were created by intensity normalization to cerebellar cortex. Regional SUVr histograms were made and images quantified using both sampling of usual average SUVr and SUVr threshold sampling of the number of voxels (NVOX) and weighted average SUVr (wSUVr) representing the extent and extent × intensity of the increased uptake, respectively. The SUVr histogram distribution in the cerebellar cortex was similar for all subjects, ranging from about 0.6-1.4. For the AD subjects, a multi-modal distribution was observed in the cortical regions, one similar to that observed in the cerebellar cortex mixed with others corresponding to increased uptake, with maximum SUVr up to 4.5 in the inferior and superior-lateral temporal cortex. Separation between populations was increased for the wSUVr and NVOX with increasing SUVr threshold, but at the expense of distorting the within-cohort spread. These preliminary data suggests that [18F]AV1451 quantification using other outcome measures than standard average regional SUVr may be useful but they would need further investigation to assess their reliability given the observed increased spread. Other reference regions or voxel-clustering strategies may prove useful in improving the accuracy of these measurements at different SUVr thresholds.

Amyloid-β25–35 induces impairment of cognitive function and long-term potentiation through phosphorylation of collapsin response mediator protein 2

Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) protein and tau deposition in the brain. Numerous studies have reported a central role of Aβ in the development of AD, but the pathogenesis is not well understood. Collapsin response mediator protein 2 (CRMP2), an intracellular protein mediating a repulsive axon guidance molecule, Semaphorin3A, is also accumulated in neurofibrillary tangles in AD brains. To gain insight into the role of CRMP2 phosphorylation in AD pathogenesis, we investigated the effects of Aβ neurotoxicity in CRMP2 phosphorylation-deficient knock-in (crmp2ki/ki) mice, in which the serine residue at 522 was replaced with alanine. Intracerebroventricular (i.c.v.) injection of Aβ25–35 peptide, a neurotoxic fragment of Aβ protein, to wild-type (wt) mice increased hippocampal phosphorylation of CRMP2. Behavioral assessment revealed that i.c.v. injection of Aβ25–35 peptide caused impairment of novel object recognition in wt mice, while the same peptide did not in crmp2ki/ki mice. In electrophysiological recording, wt and crmp2ki/ki mice have similar input–output basal synaptic transmission and paired-pulse ratios. However, long-term potentiation was impaired in hippocampal slices of Aβ25–35 peptide-treated wt but not those of crmp2ki/ki. Our findings indicate that CRMP2 phosphorylation is required for Aβ-induced impairment of cognitive memory and synaptic plasticity.

Evidence for Irreversible Inhibition of Glycogen Synthase Kinase-3β by Tideglusib

Tideglusib is a GSK-3 inhibitor currently in phase II clinical trials for the treatment of Alzheimer disease and progressive supranuclear palsy. Sustained oral administration of the compound to a variety of animal models decreases Tau hyperphosphorylation, lowers brain amyloid plaque load, improves learning and memory, and prevents neuronal loss. We report here that tideglusib inhibits GSK-3β irreversibly, as demonstrated by the lack of recovery in enzyme function after the unbound drug has been removed from the reaction medium and the fact that its dissociation rate constant is non-significantly different from zero. Such irreversibility may explain the non-competitive inhibition pattern with respect to ATP shown by tideglusib and perhaps other structurally related compounds. The replacement of Cys-199 by an Ala residue in the enzyme seems to increase the dissociation rate, although the drug retains its inhibitory activity with decreased potency and long residence time. In addition, tideglusib failed to inhibit a series of kinases that contain a Cys homologous to Cys-199 in their active site, suggesting that its inhibition of GSK-3β obeys to a specific mechanism and is not a consequence of nonspecific reactivity. Results obtained with [(35)S]tideglusib do not support unequivocally the existence of a covalent bond between the drug and GSK-3β. The irreversibility of the inhibition and the very low protein turnover rate observed for the enzyme are particularly relevant from a pharmacological perspective and could have significant implications on its therapeutic potential.

Phosphorylation of tau at Ser214 mediates its interaction with 14‐3‐3 protein: implications for the mechanism of tau aggregation

The microtubule associated protein tau is a major component of neurofibrillary tangles in Alzheimer disease brain, however the neuropathological processes behind the formation of neurofibrillary tangles are still unclear. Previously, 14-3-3 proteins were reported to bind with tau. 14-3-3 Proteins usually bind their targets through specific serine/threonine -phosphorylated motifs. Therefore, the interaction of tau with 14-3-3 mediated by phosphorylation was investigated. In this study, we show that the phosphorylation of tau by either protein kinase A (PKA) or protein kinase B (PKB) enhances the binding of tau with 14-3-3 in vitro. The affinity between tau and 14-3-3 is increased 12- to 14-fold by phosphorylation as determined by real time surface plasmon resonance studies. Mutational analyses revealed that Ser214 is critical for the phosphorylation-mediated interaction of tau with 14-3-3. Finally, in vitro aggregation assays demonstrated that phosphorylation by PKA/PKB inhibits the formation of aggregates/filaments of tau induced by 14-3-3. As the phosphorylation at Ser214 is up-regulated in fetal brain, tau's interaction with 14-3-3 may have a significant role in the organization of the microtubule cytoskeleton in development. Also as the phosphorylation at Ser214 is up-regulated in Alzheimer's disease brain, tau's interaction with 14-3-3 might be involved in the pathology of this disease.

Activated c-Jun is present in neurofibrillary tangles in Alzheimer's disease brains

Alzheimer's disease (AD) pathology is characterized by the presence of insoluble β-amyoid deposits and neurofibrillary tangles containing hyperphosphorylated tau. Increased expression of the immediate early gene product c-Jun has also been reported in post-mortem AD brains, and the presence of upstream regulators of c-Jun has been described in tangle formations. Here, we report the presence of c-Jun specifically phosphorylated on ser-63, but not ser-73, in tangle-bearing neurons and in ‘late-stage’ extracellular tangles in AD brains. Western blot analysis confirmed the presence of c-Jun phosphorylated on ser-63 but not on ser-73 in AD brain tissue. The expression of differentially phosphorylated c-Jun in the AD brain may reflect the contradictory roles of these phosphorylation sites in neurons. Furthermore, the inappropriate sequestration of phosphorylated c-Jun in tangles in AD brains may contribute to AD pathology and neurodegeneration.

Thrombin and Prothrombin Are Expressed by Neurons and Glial Cells and Accumulate in Neurofibrillary Tangles in Alzheimer Disease Brain

Thrombin is a serine protease that is generated by proteolytic cleavage of its precursor, prothrombin. We previously showed that thrombin proteolyses the microtubule-associated protein tau and that phosphorylation of tau inhibits this process. To characterize further the role of thrombin in the brain, we investigated prothrombin and thrombin expression in cultured brain cells and in brains of control, Alzheimer disease (AD) and parkinsonism-dementia complex of Guam (PDCG). We show by reverse transcriptase-polymerase chain reaction that prothrombin mRNA is expressed in brain tissues, neuroblastoma cells, and cultured human astrocytes, oligodendrocytes, and microglial cells. We also show by immunohistochemistry that the proteins prothrombin and thrombin are present in brain using specific monoclonal and polyclonal antibodies for both proteins. All antibodies stained residual serum in blood vessels, as well as normal pyramidal neurons and their processes, and some astrocytes. Additionally, in AD and PDCG cases, all antibodies stained extra- and intracellular neurofibrillary tangles (NFTs), senile plaques, and reactive microglial cells. The ubiquitous expression of prothrombin and thrombin in brain cells suggests that thrombin plays an important physiological role in normal brain. The accumulation of thrombin and prothrombin in NFTs supports the hypothesis that thrombin may be involved in tau proteolysis and that failure to metabolize tau may lead to its aggregation in neurodegenerative diseases.

Oxidative Modifications and Aggregation of Cu,Zn-Superoxide Dismutase Associated with Alzheimer and Parkinson Diseases

Although oxidative stress has been strongly implicated in the pathogenesis of Alzheimer disease (AD) and Parkinson disease (PD), the identities of specific protein targets of oxidative damage remain largely unknown. Here, we report that Cu,Zn-superoxide dismutase (SOD1), a key antioxidant enzyme whose mutations have been linked to autosomal dominant neurodegenerative disorder familial amyotrophic lateral sclerosis (ALS), is a major target of oxidative damage in AD and PD brains. By using a combination of two-dimensional gel electrophoresis, immunoblot analysis, and mass spectrometry, we have identified four human brain SOD1 isoforms with pI values of 6.3, 6.0, 5.7, and 5.0, respectively. Of these, the SOD1 pI 6.0 isoform is oxidatively modified by carbonylation, and the pI 5.0 isoform is selectively accumulated in AD and PD. Moreover, Cys-146, a cysteine residue of SOD1 that is mutated in familial ALS, is oxidized to cysteic acid in AD and PD brains. Quantitative Western blot analyses demonstrate that the total level of SOD1 isoforms is significantly increased in both AD and PD. Furthermore, immunohistochemical and double fluorescence labeling studies reveal that SOD1 forms proteinaceous aggregates that are associated with amyloid senile plaques and neurofibrillary tangles in AD brains. These findings implicate, for the first time, the involvement of oxidative damage to SOD1 in the pathogenesis of sporadic AD and PD. This work suggests that AD, PD, and ALS may share a common or overlapping pathogenic mechanism(s) that could potentially be targeted by similar therapeutic strategies.

Phosphorylation of tau by fyn: implications for Alzheimer's disease.

The abnormal phosphorylation of tau protein on serines and threonines is a hallmark characteristic of the neurofibrillary tangles of Alzheimer's disease (AD). The discovery that tau could be phosphorylated on tyrosine and evidence that Abeta signal transduction involved tyrosine phosphorylation led us to question whether tyrosine phosphorylation of tau occurred during the neurodegenerative process. In this study we determined that human tau tyr18 was phosphorylated by the src family tyrosine kinase fyn. By developing both polyclonal and monoclonal probes specific for phospho-tyr18, we found that the phosphorylation of tau at tyr18 occurred at early developmental stages in mouse but was absent in the adult. Our phosphospecific probes also revealed that paired helical filament preparations exhibited phospho-tyr18 reactivity that was sensitive to phosphotyrosine-specific protein phosphatase treatment. Moreover, immunocytochemical studies indicated that tyrosine phosphorylated tau was present in the neurofibrillary tangles in AD brain. However, the staining pattern excluded neuropil threads and dystrophic neurites indicating that tyrosine phosphorylated tau was distributed in AD brain in a manner dissimilar from other abnormally phosphorylated tau. We also found evidence suggesting that differentially phosphorylated tau existed within degenerating neurons. Our data add new support for a role for fyn in the neurodegenerative process.

Oxidative Modifications and Down-regulation of Ubiquitin Carboxyl-terminal Hydrolase L1 Associated with Idiopathic Parkinson's and Alzheimer's Diseases

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative diseases that occur either in relatively rare, familial forms or in common, sporadic forms. The genetic defects underlying several monogenic familial forms of AD and PD have recently been identified, however, the causes of other AD and PD cases, particularly sporadic cases, remain unclear. To gain insights into the pathogenic mechanisms involved in AD and PD, we used a proteomic approach to identify proteins with altered expression levels and/or oxidative modifications in idiopathic AD and PD brains. Here, we report that the protein level of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), a neuronal de-ubiquitinating enzyme whose mutation has been linked to an early-onset familial PD, is down-regulated in idiopathic PD as well as AD brains. By using a combination of two-dimensional gel electrophoresis and mass spectrometry, we have identified three human brain UCH-L1 isoforms, a full-length form and two amino-terminally truncated forms. Our proteomic analyses reveal that the full-length UCH-L1 is a major target of oxidative damage in AD and PD brains, which is extensively modified by carbonyl formation, methionine oxidation, and cysteine oxidation. Furthermore, immunohistochemical studies show that prominent UCH-L1 immunostaining is associated with neurofibrillary tangles and that the level of soluble UCH-L1 protein is inversely proportional to the number of tangles in AD brains. Together, these results provide evidence supporting a direct link between oxidative damage to the neuronal ubiquitination/de-ubiquitination machinery and the pathogenesis of sporadic AD and PD.

The p53 homologue p73 accumulates in the nucleus and localizes to neurites and neurofibrillary tangles in Alzheimer disease brain.

The molecular mechanisms that regulate neuronal survival vs. death during Alzheimer disease (AD) remain unclear. Nonetheless, a number of recent studies indicate that increased expression or altered subcellular distribution of numerous cell cycle proteins during AD may contribute to disease pathogenesis. Because homologues of p53, a key regulatory protein in the cell cycle, such as p73, have been identified and shown to participate in cellular differentiation and death pathways, we examined the expression and distribution of p73 in the hippocampus of eight control and 16 AD subjects. In control subjects, hippocampal pyramidal neurones exhibit p73 immunoreactivity that is distributed predominately in the cytoplasm. In AD hippocampus, increased levels of p73 are located in the nucleus of pyramidal neurones and p73 is located in dystrophic neurites and cytoskeletal pathology. Immunoblot analysis confirmed the presence of p73 in the hippocampus. These data indicate that p73 is expressed within hippocampal pyramidal neurones and exhibits altered subcellular distribution in AD.

Drugs targeting Alzheimer's disease: some things old and some things new.

Enormous effort is now being devoted to developing drugs that slow neurodegeneration in Alzheimer's disease (AD), although insights into AD genetics and molecular pathogenesis only arose in the last 15 years. Acetylcholinesterase inhibitors that temporarily slow loss of cognitive function remain the only approved AD drugs. Discovery of mutations in three genes leading to severe early onset AD was critical in focusing attention on the role of amyloid peptides (Abeta) in neuronal cell death, and enhanced understanding of the biology of these peptides has led to an array of mechanism-based drug discovery strategies. These include inhibitors for Abeta-generating proteases, agents that prevent or reverse Abeta oligomerization, immunotherapies to reduce Abeta in brain and plasma, and drugs to modulate cholesterol-mediated effects on Abeta transport. Strategies are also underway to minimize toxic effects of Abeta fibrils on neurons, and these include antioxidants, blockers of glutamate-mediated excitotoxicity, and modulators of inflammatory responses within the brain. Although several approaches involve new agents for recently discovered targets, many are based on new applications of existing drugs such as statins and nonsteroidal anti-inflammatory drugs. Discovery of abnormally phosphorylated tau protein in neurofibrillary tangles in AD brain has led to strategies for identifying selective inhibitors of tau kinases and central nervous system/brain-permeable drugs that help maintain microtubule integrity. Clearly, a large gap exists between our understanding of the cellular cascades targeted in drug discovery and widespread failure of the nervous system that AD represents. Nevertheless, the pace of recent research clearly supports optimism that slowing progression of AD will soon be possible.

Phorbol ester enhances phosphorylated tau protein immunoreactivity in neuronal cultures

One of the hallmarks of Alzheimer's disease (AD) is neurofibrillary degeneration which results from the aggregation of phosphorylated tau proteins into paired helical filament (PHF) structures. AD 2 is a new monoclonal antibody raised against PHF tau which detects neurofibrillary tangles in AD brain. In primary neuronal cultures, phorbol ester treatment induced a time- and dose-dependent increase in AD 2 immunoreactivity quantified by laser confocal microscopy and immunoblottings. Alkaline phosphatase treatment reversed these immunocytochemical changes. These results suggest that the modifications of neuronal metabolism induced by phorbol ester including protein kinase C activation produce an increase in phosphorylated tau immunoreactivity.

Midkine, a Novel Neurotrophic Factor, Is Present in Senile Plaques of Alzheimer Disease

An affinity purified antibody specific for midkine (MK) stained senile plaques in the brain of Alzheimer disease (AD) patients. After formic acid treatment, plaque staining was dramatically enhanced, and almost all β-amyloid protein (BAP) deposits were also immunoreactive for MK. MK-immunoreactivity was not observed in normal cellular components nor in other pathological lesions including tangles in AD brain. Control brain sections were not immunoreactive for MK. The presence of MK in AD brain but not in control brain was confirmed by Western blotting. MK appears to be involved in the pathological process leading to senile plaque formation.

Pathologic Changes in Olfactory Neurons in Alzheimer's Diseasea

Olfactory deficits and degenerative changes in central olfactory pathways are prominent in patients with Alzheimer's disease (AD). We hypothesized that peripheral olfactory neurons that reside in the nasal epithelium would show degenerative changes similar to the characteristic pathologic features of AD brain. Immunohistochemical studies of nasal tissue taken at autopsy reveal extensive degeneration in the sensory epithelium as well as abnormal neurites that share immunoreactive epitopes with dystrophic neurites and neurofibrillary tangles of the AD brain. The neuritic masses are stained with well-characterized monoclonal antibodies that do not normally stain olfactory neurons but which are very reactive with dystrophic neuritic structures and neurofibrillary tangles in AD brain. These include antibodies to phosphorylated and nonphosphorylated neurofilament subunits, tau, and also ALZ50, which is characteristically reactive with AD but not with normal brains. Such changes are present in 81% of AD patients. Similar accumulations of ectopic neurites are found in the olfactory epithelium of about 22% of non-demented patients. Preliminary statistical analysis fails to reveal any age-linked association. It has been proposed that the aged monkey is a good model for AD inasmuch as amyloid accumulations similar to those of humans are found in monkey brain. We examined a series of 13 rhesus monkeys, including aged animals with behavioral deficits. Although the olfactory epithelium was very similar to that of humans, no abnormal olfactory structures were observed. Aged rhesus monkeys do not appear to be a good model for the neuritic abnormalities of AD.

Anomalous accumulation of τ and ubiquitin immunoreactivities in rat brain caused by protease inhibition and by normal aging: a clue to PHF pathogenesis?

Rats received intraventricular infusion of leupeptin or saline and brain sections were immunostained with antibodies to τ (anti-HFoPHF) or ubiquitin. Results were compared with immunostaining on normal aged rat brains and Alzheimer's disease (AD) brains. Both antibodies stained Purkinje cell perikarya and dendrites of leupeptin (bu not salin)-treated and aged rat brains, as well as senile plaque neurites and neurofibrillary tangles in AD brains. The results are consistent with the hypothesis that paired helical filament (PHF) formation involves defective protein degradation.