Neurons In Alzheimer's Disease Brain Research Articles

Evidence that Alzheimer's Disease Is a Disease of Competitive Synaptic Plasticity Gone Awry.

Mounting evidence indicates that a physiological function of amyloid-β (Aβ) is to mediate neural activity-dependent homeostatic and competitive synaptic plasticity in the brain. I have previously summarized the lines of evidence supporting this hypothesis and highlighted the similarities between Aβ and anti-microbial peptides in mediating cell/synapse competition. In cell competition, anti-microbial peptides deploy a multitude of mechanisms to ensure both self-protection and competitor elimination. Here I review recent studies showing that similar mechanisms are at play in Aβ-mediated synapse competition and perturbations in these mechanisms underpin Alzheimer's disease (AD). Specifically, I discuss evidence that Aβ and ApoE, two crucial players in AD, co-operate in the regulation of synapse competition. Glial ApoE promotes self-protection by increasing the production of trophic monomeric Aβ and inhibiting its assembly into toxic oligomers. Conversely, Aβ oligomers, once assembled, promote the elimination of competitor synapses via direct toxic activity and amplification of "eat-me" signals promoting the elimination of weak synapses. I further summarize evidence that neuronal ApoE may be part of a gene regulatory network that normally promotes competitive plasticity, explaining the selective vulnerability of ApoE expressing neurons in AD brains. Lastly, I discuss evidence that sleep may be key to Aβ-orchestrated plasticity, in which sleep is not only induced by Aβ but is also required for Aβ-mediated plasticity, underlining the link between sleep and AD. Together, these results strongly argue that AD is a disease of competitive synaptic plasticity gone awry, a novel perspective that may promote AD research.

Monoamine oxidase B is elevated in Alzheimer disease neurons, is associated with \u03b3-secretase and regulates neuronal amyloid \u03b2-peptide levels

BackgroundIncreased levels of the pathogenic amyloid β-peptide (Aβ), released from its precursor by the transmembrane protease γ-secretase, are found in Alzheimer disease (AD) brains. Interestingly, monoamine oxidase B (MAO-B) activity is also increased in AD brain, but its role in AD pathogenesis is not known. Recent neuroimaging studies have shown that the increased MAO-B expression in AD brain starts several years before the onset of the disease. Here, we show a potential connection between MAO-B, γ-secretase and Aβ in neurons.MethodsMAO-B immunohistochemistry was performed on postmortem human brain. Affinity purification of γ-secretase followed by mass spectrometry was used for unbiased identification of γ-secretase-associated proteins. The association of MAO-B with γ-secretase was studied by coimmunoprecipitation from brain homogenate, and by in-situ proximity ligation assay (PLA) in neurons as well as mouse and human brain sections. The effect of MAO-B on Aβ production and Notch processing in cell cultures was analyzed by siRNA silencing or overexpression experiments followed by ELISA, western blot or FRET analysis. Methodology for measuring relative intraneuronal MAO-B and Aβ42 levels in single cells was developed by combining immunocytochemistry and confocal microscopy with quantitative image analysis.ResultsImmunohistochemistry revealed MAO-B staining in neurons in the frontal cortex, hippocampus CA1 and entorhinal cortex in postmortem human brain. Interestingly, the neuronal staining intensity was higher in AD brain than in control brain in these regions. Mass spectrometric data from affinity purified γ-secretase suggested that MAO-B is a γ-secretase-associated protein, which was confirmed by immunoprecipitation and PLA, and a neuronal location of the interaction was shown. Strikingly, intraneuronal Aβ42 levels correlated with MAO-B levels, and siRNA silencing of MAO-B resulted in significantly reduced levels of intraneuronal Aβ42. Furthermore, overexpression of MAO-B enhanced Aβ production.ConclusionsThis study shows that MAO-B levels are increased not only in astrocytes but also in pyramidal neurons in AD brain. The study also suggests that MAO-B regulates Aβ production in neurons via γ-secretase and thereby provides a key to understanding the relationship between MAO-B and AD pathogenesis. Potentially, the γ-secretase/MAO-B association may be a target for reducing Aβ levels using protein–protein interaction breakers.

Caspase-cleaved Tau-D421 is colocalized with the immunophilin FKBP52 in the autophagy-endolysosomal system of Alzheimer's disease neurons

Pathologic modifications of the Tau protein leading to neurofibrillary tangle (NFT) formation are a common feature of a wide range of neurodegenerative diseases known as tauopathies, which include Alzheimer's disease (AD). We previously showed that the immunophilin FKBP52 physically and functionally interacts with Tau, and we recently reported that FKBP52 levels are abnormally low in AD patients' brains. To decipher the mechanism of FKBP52 decrease in AD brains, we performed multiple labeling immunohistofluorescence and lysosomal purification using postmortem brain samples of healthy controls (n = 8) and AD (n = 20) patients. Confocal analysis revealed that FKBP52 localizes to the endolysosomal system. We also report FKBP52 colocalization with the truncated Tau-D421 in the autophagy-endolysosomal system in some AD neurons and that the decrease of FKBP52 correlates with NFT formation. Additional experiments of autophagy inhibition in Tau-inducible SH-SY5Y cells allowed demonstrating FKBP52 release in the extracellular milieu. Our findings point out the possibility that FKBP52 could be abnormally released from NFTs negative neurons in AD brains in correlation with the early pathologic Tau-D421 neuronal accumulation.

Mammalian Target of Rapamycin (mTor) Mediates Tau Protein Dyshomeostasis: IMPLICATION FOR ALZHEIMER DISEASE

Previous evidence from post-mortem Alzheimer disease (AD) brains and drug (especially rapamycin)-oriented in vitro and in vivo models implicated an aberrant accumulation of the mammalian target of rapamycin (mTor) in tangle-bearing neurons in AD brains and its role in the formation of abnormally hyperphosphorylated tau. Compelling evidence indicated that the sequential molecular events such as the synthesis and phosphorylation of tau can be regulated through p70 S6 kinase, the well characterized immediate downstream target of mTor. In the present study, we further identified that the active form of mTor per se accumulates in tangle-bearing neurons, particularly those at early stages in AD brains. By using mass spectrometry and Western blotting, we identified three phosphoepitopes of tau directly phosphorylated by mTor. We have developed a variety of stable cell lines with genetic modification of mTor activity using SH-SY5Y neuroblastoma cells as background. In these cellular systems, we not only confirmed the tau phosphorylation sites found in vitro but also found that mTor mediates the synthesis and aggregation of tau, resulting in compromised microtubule stability. Changes of mTor activity cause fluctuation of the level of a battery of tau kinases such as protein kinase A, v-Akt murine thymoma viral oncogene homolog-1, glycogen synthase kinase 3β, cyclin-dependent kinase 5, and tau protein phosphatase 2A. These results implicate mTor in promoting an imbalance of tau homeostasis, a condition required for neurons to maintain physiological function.

Intracellular Accumulation of Toxic Turn Amyloid-β is Associated with Endoplasmic Reticulum Stress in Alzheimer's Disease

Amyloid-β protein (Aβ) accumulates in the neurons of Alzheimer's disease (AD) patients at an early stage of the disease. Recently, we found that Aβ with a toxic turn at positions 22 and 23 accumulates in neurons in AD brain. Here, we studied the accumulation of Aβ, toxic turn Aβ and high-molecular-weight Aβ oligomers in presenilin 1 (PS1) gene-transfected SH-SY5Y cells as well as in the brains of 3xTg-AD mice and AD patients. Immunostaining revealed that accumulation of toxic turn Aβ was promoted in G384A- and I143T-mutant PS1-transfected cells and further enhanced by co-transfection of cells with the Aβ-precursor protein (AβPP) gene. In contrast, accumulation of high-molecular-weight Aβ oligomers was promoted in mutant PS1 cells but attenuated by co-transfection of cells with the AβPP gene. Toxic turn Aβ was detected in the neurons of 3xTg-AD mice aged 2 months, when the mice were cognitively unimpaired. In contrast, high-molecular-weight Aβ oligomers were detected in the neurons of 7-month-old mice, when memory dysfunction is apparent. Furthermore, immunostaining and western blotting for Rab4, Rab6 and GRP78 revealed increased levels of these proteins in mutant PS1 cells and their accumulation in the neurons of 3xTg-AD mice. Remarkably, GRP78 immunoreactivity was increased at 2 months of age. Double-label immunostaining of AD brain revealed an apparent association between toxic turn Aβ and GRP78, an endoplasmic reticulum (ER) stress marker. Intraneuronal accumulation of toxic turn Aβ may be associated with ER stress in the brains of AD model mice and AD patients at an early stage.

P2‐006: Increased cerebrospinal fluid levels of double‐stranded RNA‐dependant protein kinase in Alzheimer's disease

Background The pathological hallmarks of Alzheimer's disease (AD) include accumulation of amyloid-β (As) peptide forming extracellular senile plaques, neurofibrillary tangles made of hyperphosphorylated tau protein with neuronal loss. Aβ peptide (1–42), total tau (T-tau), and phosphorylated tau at threonine 181 (p181tau) levels in the cerebrospinal fluid (CSF) are now validated biomarkers. The proapoptotic kinase R (PKR), is activated by Aβ accumulates in degenerating neurons in AD brains and controls protein synthesis and indirectly tau phosphorylation. Methods In a prospective cohort study, the CSF of 91 patients were studied (AD: 45; amnestic mild cognitive impairment: 11; neurological disease control subjects [NDC]: 35). The levels of total PKR (T-PKR), phosphorylated PKR (pPKR), As 1–42, T-tau, and p181tau were assessed by immunoblotting or enzyme-linked immunosorbent assay methods. Receivers operating characteristic curves were used to examine the discriminatory power of T-PKR, pPKR, and pPKR/T-PKR ratio between AD and NDC patients. Results Total PKR and pPKR concentrations were elevated in AD and amnestic mild cognitive impairment subjects. We have determined a pPKR value (optical density units) that could discriminate AD patients from control subjects with a sensitivity of 91.1% and a specificity of 94.3%. Among AD patients, T-PKR and pPKR levels correlate with CSF p181tau levels. Some AD patients with normal CSF As, T-tau, or p181tau levels had abnormal T-PKR and pPKR levels. Conclusions The evaluation of CSF T-PKR and pPKR can discriminate between AD patients and NDC and could help to improve the biochemical diagnosis of AD.

Heparan Sulfate Subdomains that are Degraded by Sulf Accumulate in Cerebral Amyloid ß Plaques of Alzheimer's Disease: Evidence from Mouse Models and Patients

Alzheimer's disease (AD) is characterized by extracellular cerebral accumulation of amyloid β peptide (Aβ). Heparan sulfate (HS) is a glycosaminoglycan that is abundant in the extracellular space. The state of sulfation within the HS chain influences its ability to interact with a variety of proteins. Highly sulfated domains within HS are crucial for Aβ aggregation in vitro. Here, we investigated the expression of the sulfated domains and HS disaccharide composition in the brains of Tg2576, J20, and T41 transgenic AD mouse models, and patients with AD. RB4CD12, a phage display antibody, recognizes highly sulfated domains of HS. The RB4CD12 epitope is abundant in the basement membrane of brain vessels under physiological conditions. In the cortex and hippocampus of the mice and patients with AD, RB4CD12 strongly stained both diffuse and neuritic amyloid plaques. Interestingly, RB4CD12 also stained the intracellular granules of certain hippocampal neurons in AD brains. Disaccharide compositions in vessel-enriched and nonvasculature fractions of Tg2576 mice and AD patients were found to be comparable to those of non-transgenic and non-demented controls, respectively. The RB4CD12 epitope in amyloid plaques was substantially degraded ex vivo by Sulf-1 and Sulf-2, extracellular HS endosulfatases. These results indicate that formation of highly sulfated HS domains may be upregulated in conjunction with AD pathogenesis, and that these domains can be enzymatically remodeled in AD brains.

Increased Cerebrospinal Fluid Levels of Double-Stranded RNA-Dependant Protein Kinase in Alzheimer's Disease

The pathological hallmarks of Alzheimer's disease (AD) include accumulation of amyloid-β (Aß) peptide forming extracellular senile plaques, neurofibrillary tangles made of hyperphosphorylated tau protein with neuronal loss. Aβ peptide (1-42), total tau (T-tau), and phosphorylated tau at threonine 181 (p181tau) levels in the cerebrospinal fluid (CSF) are now validated biomarkers. The proapoptotic kinase R (PKR), is activated by Aβ accumulates in degenerating neurons in AD brains and controls protein synthesis and indirectly tau phosphorylation. In a prospective cohort study, the CSF of 91 patients were studied (AD: 45; amnestic mild cognitive impairment: 11; neurological disease control subjects [NDC]: 35). The levels of total PKR (T-PKR), phosphorylated PKR (pPKR), Aß 1-42, T-tau, and p181tau were assessed by immunoblotting or enzyme-linked immunosorbent assay methods. Receivers operating characteristic curves were used to examine the discriminatory power of T-PKR, pPKR, and pPKR/T-PKR ratio between AD and NDC patients. Total PKR and pPKR concentrations were elevated in AD and amnestic mild cognitive impairment subjects. We have determined a pPKR value (optical density units) that could discriminate AD patients from control subjects with a sensitivity of 91.1% and a specificity of 94.3%. Among AD patients, T-PKR and pPKR levels correlate with CSF p181tau levels. Some AD patients with normal CSF Aß, T-tau, or p181tau levels had abnormal T-PKR and pPKR levels. The evaluation of CSF T-PKR and pPKR can discriminate between AD patients and NDC and could help to improve the biochemical diagnosis of AD.

The Pathological Roles of Ganglioside Metabolism in Alzheimer's Disease: Effects of Gangliosides on Neurogenesis

Conversion of the soluble, nontoxic amyloid β-protein (Aβ) into an aggregated, toxic form rich in β-sheets is a key step in the onset of Alzheimer's disease (AD). It has been suggested that Aβ induces changes in neuronal membrane fluidity as a result of its interactions with membrane components such as cholesterol, phospholipids, and gangliosides. Gangliosides are known to bind Aβ. A complex of GM1 and Aβ, termed “GAβ”, has been identified in AD brains. Abnormal ganglioside metabolism also may occur in AD brains. We have reported an increase of Chol-1α antigens, GQ1bα and GT1aα, in the brain of transgenic mouse AD model. GQ1bα and GT1aα exhibit high affinities to Aβs. The presence of Chol-1α gangliosides represents evidence for genesis of cholinergic neurons in AD brains. We evaluated the effects of GM1 and Aβ1–40 on mouse neuroepithelial cells. Treatment of these cells simultaneously with GM1 and Aβ1–40 caused a significant reduction of cell number, suggesting that Aβ1–40 and GM1 cooperatively exert a cytotoxic effect on neuroepithelial cells. An understanding of the mechanism on the interaction of GM1 and Aβs in AD may contribute to the development of new neuroregenerative therapies for this disorder.

I2PP2A regulates p53 and Akt correlatively and leads the neurons to abort apoptosis

A chronic neuron loss is the cardinal pathology in Alzheimer disease (AD), but it is still not understood why most neurons in AD brain do not accomplish apoptosis even though they are actually exposed to an environment with enriched proapoptotic factors. Protein phosphatase-2A inhibitor-2 (I(2)(PP2A)), an endogenous PP2A inhibitor, is significantly increased in AD brain, but the role of I(2)(PP2A) in AD-like neuron loss is elusive. Here, we show that I(2)(PP2A) regulates p53 and Akt correlatively. The mechanisms involve activated transcription and p38 MAPK activities. More importantly, we demonstrate that the simultaneous activation of Akt induced by I(2)(PP2A) counteracts the hyperactivated p53-induced cell apoptosis. Furthermore, I(2)(PP2A), p53 and Akt are all elevated in the brain of mouse model and AD patients. Our results suggest that the increased I(2)(PP2A) may trigger apoptosis by p53 upregulation, but due to simultaneous activation of Akt, the neurons are aborted from the apoptotic pathway. This finding contributes to the understanding of why most neurons in AD brain do not undergo apoptosis.

Interaction of Double-stranded RNA-dependent Protein Kinase (PKR) with the Death Receptor Signaling Pathway in Amyloid β (Aβ)-treated Cells and in APPSLPS1 Knock-in Mice

For 10 years, research has focused on signaling pathways controlling translation to explain neuronal death in Alzheimer Disease (AD). Previous studies demonstrated in different cellular and animal models and AD patients that translation is down-regulated by the activation of double-stranded RNA-dependent protein kinase (PKR). Among downstream factors of PKR, the Fas-associated protein with a death domain (FADD) and subsequent activated caspase-8 are responsible for PKR-induced apoptosis in recombinant virus-infected cells. However, no studies have reported the role of PKR in death receptor signaling in AD. The aim of this project is to determine physical and functional interactions of PKR with FADD in amyloid-beta peptide (Abeta) neurotoxicity and in APP(SL)PS1 KI transgenic mice. In SH-SY5Y cells, results showed that Abeta42 induced a large increase in phosphorylated PKR and FADD levels and a physical interaction between PKR and FADD in the nucleus, also observed in the cortex of APP(SL)PS1 KI mice. However, PKR gene silencing or treatment with a specific PKR inhibitor significantly prevented the increase in pT(451)-PKR and pS(194)-FADD levels in SH-SY5Y nuclei and completely inhibited activities of caspase-3 and -8. The contribution of PKR in neurodegeneration through the death receptor signaling pathway may support the development of therapeutics targeting PKR to limit neuronal death in AD.

The Excitotoxin Quinolinic Acid Induces Tau Phosphorylation in Human Neurons

Some of the tryptophan catabolites produced through the kynurenine pathway (KP), and more particularly the excitotoxin quinolinic acid (QA), are likely to play a role in the pathogenesis of Alzheimer's disease (AD). We have previously shown that the KP is over activated in AD brain and that QA accumulates in amyloid plaques and within dystrophic neurons. We hypothesized that QA in pathophysiological concentrations affects tau phosphorylation. Using immunohistochemistry, we found that QA is co-localized with hyperphosphorylated tau (HPT) within cortical neurons in AD brain. We then investigated in vitro the effects of QA at various pathophysiological concentrations on tau phosphorylation in primary cultures of human neurons. Using western blot, we found that QA treatment increased the phosphorylation of tau at serine 199/202, threonine 231 and serine 396/404 in a dose dependent manner. Increased accumulation of phosphorylated tau was also confirmed by immunocytochemistry. This increase in tau phosphorylation was paralleled by a substantial decrease in the total protein phosphatase activity. A substantial decrease in PP2A expression and modest decrease in PP1 expression were observed in neuronal cultures treated with QA. These data clearly demonstrate that QA can induce tau phosphorylation at residues present in the PHF in the AD brain. To induce tau phosphorylation, QA appears to act through NMDA receptor activation similar to other agonists, glutamate and NMDA. The QA effect was abrogated by the NMDA receptor antagonist memantine. Using PCR arrays, we found that QA significantly induces 10 genes in human neurons all known to be associated with AD pathology. Of these 10 genes, 6 belong to pathways involved in tau phosphorylation and 4 of them in neuroprotection. Altogether these results indicate a likely role of QA in the AD pathology through promotion of tau phosphorylation. Understanding the mechanism of the neurotoxic effects of QA is essential in developing novel therapeutic strategies for AD.

TDP-43: A reliable immunohistochemistry marker for inclusion body myositis?

TDP-43 is a 414 amino acid protein encoded by the transactive response (TAR) DNA binding protein gene on chromosome 1p36. It was first cloned as a human protein capable of binding to the TAR DNA of HIV-1.11 It was later identified as part of a complex involved in splicing of the cystic fibrosis transmembrane conductance regulator gene.3 TDP-43 is highly conserved, ubiquitously expressed and predominantly localized to the nucleus under normal conditions. It consists of two RNA recognition motifs and a glycine-rich C-terminal region which interacts with other members of the heterogeneous ribonucleoprotein (hnRNP) family.2,5 TDP-43 is chiefly involved in well characterized transcription function and RNA splicing regulation. Recent studies suggest that TDP-43 may be involved in other cellular processes, such as microRNA biogenesis, apoptosis, cell division, mRNA stabilization and regulation of neuronal plasticity.2,5 Since its link to sporadic amyotrophic lateral sclerosis (sALS), frontotemporal dementia (FTD) with ubiquitinated protein inclusions (UBIs) (FTD-U) and FTD-U/ALS in 2006, overexpression of TDP-43 has been reported in other conditions with cellular overexpression, including Guaminian ALS-parkinsonism-dementia complex, progranulin gene mutation, VCP (Valosin-containing protein) gene mutation, dementia linked to chromosome 9 without VCP gene mutation, sporadic and hereditary IBM, and myofibrillar and desmin myopathies.6,7,9,12,14,15 Earlier this year, two independent researchers8,16 reported that mutations in the gene for another DNA/RNA binding protein, fused in sarcoma or translocation in liposarcoma (FUS/TLS), also trigger UBIs and premature degeneration of motor neurons. FUS/TLS protein has not yet been studied in IBM. Nevertheless, alteration in RNA processing and protein aggregates appears to be the key event in these motor neuron and muscle diseases. Sporadic inclusion body myositis (sIBM) is a common form of inflammatory muscle disease in older patients. In the first detailed pathological report of sIBM,4 Carpenter et al. commented on the characteristic inclusions as well as the nuclear degenerative process. Subsequent investigations (reviewed by Oldfors and Lindberg10) revealed similar inclusions – UBIs in both dominant and recessive forms of hereditary IBM (hIBM), oculopharyngeal muscular dystrophy, and in IBM with Paget disease of bone and frontotemporal dementia. Interestingly, when protein epitopes of UBIs in muscle fibers of sIBM were further investigated, it became clear that several “alien” molecules were overexpressed. These included -amyloid, phosphorylated tau and SMI-31 that incidentally were also the constituents of UBIs in the neurons of Alzheimer disease brains. These observations prompted Banwell and Engel1 to search for other protein molecules that were overexpressed in astrocytes and astroglial cells in the vicinity of senile plaques of Alzheimer disease brains. One such molecule Bcrystallin, indeed, was found to be overexpressed in sIBM.1 The demonstration of either immunoreactivity with antibodies to ubiquitin, SMI-31 or B-crystallin, or the finding of Congo red fluorescence ( -amyloid) then became the standard procedures for the histological diagnosis of IBM. Over the past year, TDP-43 immunoreactive UBIs have been reported in sIBM7,15 and in familial myopathies with or without vacuolar change.7,12,15 In this issue of Muscle and Nerve, Salajegheh and colleagues13 confirm and extend the recent observations7,12,15 of excessive TDP-43 in IBM muscle fibers. These findings are important on several counts. First, the authors found an average of nearly 1 in 4 Correspondence to: Rup Tandan, e-mail: rup.tandan@uvm.edu

Hippocampal neurogenesis during normal and pathological aging

It is now widely accepted that new neurons continue to be added to the brain throughout life including during normal aging. The finding of adult neurogenesis in the hippocampus, a structure involved in the processing of memories, has favored the idea that newborn neurons might subserve cognitive functions. Recent work on human post-mortem tissues and mice models of Alzheimer's disease (AD) has reported persistent hippocampal proliferative capacity during pathological aging. Although it is not yet clear whether neurogenesis leads to the production of fully functional mature neurons in AD brains, these findings open prospects for cell-replacement therapies. Strategies aimed at promoting neurogenesis may also contribute to improve cognitive deficits caused by normal or pathological aging.

C‐Jun phosphorylation in Alzheimer disease

The c-Jun N-terminal kinase (JNK) pathway is known to be activated by oxidative stress and can lead to either defensive-protective adaptations in the cell or apoptosis. The JNK pathway is activated in Alzheimer disease (AD), as demonstrated in studies showing higher levels of phospho-JNK in affected neurons in AD brains than in controls. c-Jun, a transcription factor, is the downstream effector of JNK, whose activation requires phosphorylation of Ser63/Ser73. In this study, we characterized and compared the localization of c-Jun phosphorylated at either Ser63 or Ser73 in the hippocampi of AD cases with that in age-matched controls. Phospho-c-Jun (Ser73) was found to be strongly associated with neurofibrillary tangles and granulovacuolar degeneration (GVD) in addition to the nuclei in neurons in the hippocampal regions of the AD brain, but was virtually absent in most controls. Phospho-c-Jun (Ser63) was also found to be associated with GVD in AD brains. Indeed, phospho-c-Jun (Ser73) immunostaining was much more extensive than that of phospho-c-Jun (Ser63), with all the phospho-c-Jun (Ser63)-positive neurons also being phospho-c-Jun (Ser73) positive. Significant overlap between phospho-c-Jun and phospho-JNK suggested a mechanistic link. In addition, the neurons showing increased levels of phospho-c-Jun (Ser73) in the cytoplasmic GVD were negative for TUNEL, suggesting a mechanism protecting the cells from death. Overall, this study demonstrated specific alterations in c-Jun phosphorylation and distribution in AD which is not necessarily linked to apoptosis but rather may represent an adaptation process in the face of oxidative stress.

Targeting the Role of the Endosome in the Pathophysiology of Alzheimer's Disease: A Strategy for Treatment

Membrane-bound endosomal vesicles play an integral role in multiple cellular events, including protein processing and turnover, and often critically regulate the cell-surface availability of receptors and other plasma membrane proteins in many different cell types. Neurons are no exception, being dependent on endosomal function for housekeeping and synaptic events. Growing evidence suggests a link between neuronal endosomal function and Alzheimer's disease (AD) pathophysiology. Endosomal abnormalities invariably occur within neurons in AD brains, and endocytic compartments are one likely site for the production of the pathogenic beta-amyloid peptide (Abeta), which accumulates within the brain during the disease and is generated by proteolytic processing of the amyloid precursor protein (APP). The enzymes and events involved in APP processing are appealing targets for therapeutic agents aimed at slowing or reversing the pathogenesis of AD. The neuronal endosome may well prove to be the intracellular site of action for inhibitors of beta-amyloidogenic APP processing. We present here the view that knowledge of the endosomal system in the disease can guide drug discovery of AD therapeutic agents.

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.

Ribosomal RNA in Alzheimer Disease Is Oxidized by Bound Redox-active Iron

Oxidative modification of cytoplasmic RNA in vulnerable neurons is an important, well documented feature of the pathophysiology of Alzheimer disease. Here we report that RNA-bound iron plays a pivotal role for RNA oxidation in vulnerable neurons in Alzheimer disease brain. The cytoplasm of hippocampal neurons showed significantly higher redox activity and iron(II) staining than age-matched controls. Notably, both were susceptible to RNase, suggesting a physical association of iron(II) with RNA. Ultrastructural analysis further suggested an endoplasmic reticulum association. Both rRNA and mRNA showed twice the iron binding as tRNA. rRNA, extremely abundant in neurons, was considered to provide the greatest number of iron binding sites among cytoplasmic RNA species. Interestingly, the difference of iron binding capacity disappeared after denaturation of RNA, suggesting that the higher order structure may contribute to the greater iron binding of rRNA. Reflecting the difference of iron binding capacity, oxidation of rRNA by the Fenton reaction formed 13 times more 8-hydroxyguanosine than tRNA. Consistent with in situ findings, ribosomes purified from Alzheimer hippocampus contained significantly higher levels of RNase-sensitive iron(II) and redox activity than control. Furthermore, only Alzheimer rRNA contains 8-hydroxyguanosine in reverse transcriptase-PCR. Addressing the biological significance of ribosome oxidation by redox-active iron, in vitro translation with oxidized ribosomes from rabbit reticulocyte showed a significant reduction of protein synthesis. In conclusion these results suggest that rRNA provides a binding site for redox-active iron and serves as a redox center within the cytoplasm of vulnerable neurons in Alzheimer disease in advance of the appearance of morphological change indicating neurodegeneration.

Mitochondrial alterations in Alzheimer's disease.

Morphological alterations of mitochondria may be related to metabolic and energy deficiency in neurons in Alzheimer's disease (AD) and other neurodegenerative disorders. In previous studies on the morphological and morphometric estimation of mitochondria in AD electron microscopy revealed substantial morphological and morphometric changes in the hippocampus, the acoustic cortex, the frontal cortex, and the cerebellum. This study extends this observation to subcortical centers, namely the thalamus, the globus pallidus, the red nucleus, and the locus caeruleus in 10 brains of patients who suffered from AD. The morphological alterations consisted of very obvious changes of the mitochondrial cristae, accumulation of osmiophilic material and decrease of their size, in comparison with the normal controls. Mitochondrial alterations were particularly prominent in neurons, which showed loss of dendritic spines and abbreviation of the dendritic arborization. The ultrastructural study of a large number of neurons in the thalamus and the red nucleus revealed that the mitochondrial alterations did not coexist with cytoskeletal pathology and accumulation of amyloid deposits. However, they were prominent in neurons, which demonstrated fragmentation of the cisternae of the Golgi apparatus. The morphological alterations of the mitochondria presumably suggest oxidative damage in neurons in AD brains.

"Emerging Alzheimer's disease therapies: focusing on the future".

The Center for Neurodegenerative Disease Research (CNDR) organized a 1 day symposium entitled "Emerging Alzheimer's disease Therapies: Focusing On The Future" on November 7th, 2001 at the University of Pennsylvania in Philadelphia, PA. The agenda (Fig. 1) focused on novel therapies for Alzheimer's disease (AD) designed to prevent/eliminate Abeta deposits in the brains of AD patients. While fibrillar Abeta deposits known as senile plaques (SPs) and intraneuronal tau fibrils known as neurofibrillary tangles (NFTs) are diagnostic of AD, >50% of patients with familial or sporadic AD as well as elderly Down's syndrome patients with AD harbor a third type of brain amyloid known as Lewy bodies formed by intraneuronal alpha-synuclein fibrils. Thus, AD is a "triple brain amyloidosis" since three different proteins (tau, alpha-synuclein) or peptide fragments (Abeta) of a larger Abeta precursor protein (APP) fibrillize and aggregate into pathological deposits of amyloid within (NFTs, LBs) and outside (SPs) neurons in AD brains. The symposium is summarized here followed by reviews from symposium speakers who describe potential anti-Abeta therapies some of which are in clinical trials.