Increased occurrence of protein kinase CK2 in astrocytes in Alzheimer's disease pathology.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disease. In addition to the occurrence of amyloid deposits and widespread tau pathology, AD is associated with a neuroinflammatory response characterized by the activation of microglia and astrocytes. Protein kinase 2 (CK2, former casein kinase II) is involved in a wide variety of cellular processes. Previous studies on CK2 in AD showed controversial results, and the involvement of CK2 in neuroinflammation in AD remains elusive. METHODS: In this study, we used immunohistochemical and immunofluorescent staining methods to investigate the localization of CK2 in the hippocampus and temporal cortex of patients with AD and non-demented controls. We compared protein levels with Western blotting analysis, and we investigated CK2 activity in human U373 astrocytoma cells and human primary adult astrocytes stimulated with IL-1ß or TNF-α. RESULTS: We report increased levels of CK2 in the hippocampus and temporal cortex of AD patients compared to non-demented controls. Immunohistochemical analysis shows CK2 immunoreactivity in astrocytes in AD and control cases. In AD, the presence of CK2 immunoreactive astrocytes is increased. CK2 immunopositive astrocytes are associated with amyloid deposits, suggesting an involvement of CK2 in the neuroinflammatory response. In U373 cells and human primary astrocytes, the selective CK2 inhibitor CX-4945 shows a dose-dependent reduction of the IL-1ß or TNF-α induced MCP-1 and IL-6 secretion. CONCLUSIONS: This data suggests that CK2 in astrocytes is involved in the neuroinflammatory response in AD. The reduction in pro-inflammatory cytokine secretion by human astrocytes using the selective CK2 inhibitor CX-4945 indicates that CK2 could be a potential target to modulate neuroinflammation in AD.

Profiling the human hippocampal proteome at all pathologic stages of Alzheimer's disease.

INTRODUCTION: We performed a comprehensive quantitative proteomics study on human hippocampus tissue involving all Braak stages to assess changes in protein abundance over the various stages of Alzheimer's disease (AD). METHODS: Hippocampal subareas CA1 and subiculum of 40 cases were isolated using laser capture microdissection and analyzed using mass spectrometry. Immunoblotting and immunohistochemistry were used for validation. RESULTS: Over the Braak stages, an altered expression was found for 372 proteins including changes in levels of extracellular matrix components, and in calcium-dependent signaling proteins. Early changes were observed in levels of proteins related to cytoskeletal dynamics and synaptic components including an increase in RIMS1 and GRIK4. Several synaptic proteins, such as BSN, LIN7A, DLG2, -3, and -4, exhibit an early-up, late-down expression pattern. DISCUSSION: This study provides new insight into AD-dependent changes in protein levels in the hippocampus during AD pathology, identifying potential novel therapeutic targets and biomarkers.

Proliferation in the Alzheimer hippocampus is due to microglia, not astroglia, and occurs at sites of amyloid deposition.

Microglia and astrocytes contribute to Alzheimer's disease (AD) etiology and may mediate early neuroinflammatory responses. Despite their possible role in disease progression and despite the fact that they can respond to amyloid deposition in model systems, little is known about whether astro- or microglia can undergo proliferation in AD and whether this is related to the clinical symptoms or to local neuropathological changes. Previously, proliferation was found to be increased in glia-rich regions of the presenile hippocampus. Since their phenotype was unknown, we here used two novel triple-immunohistochemical protocols to study proliferation in astro- or microglia in relation to amyloid pathology. We selected different age-matched cohorts to study whether proliferative changes relate to clinical severity or to neuropathological changes. Proliferating cells were found across the hippocampus but never in mature neurons or astrocytes. Almost all proliferating cells were co-labeled with Iba1+, indicating that particularly microglia contribute to proliferation in AD. Proliferating Iba1+ cells was specifically seen within the borders of amyloid plaques, indicative of an active involvement in, or response to, plaque accumulation. Thus, consistent with animal studies, proliferation in the AD hippocampus is due to microglia, occurs in close proximity of plaque pathology, and may contribute to the neuroinflammation common in AD.

Ubiquilin 2 is not associated with tau pathology.

Accumulation of aberrant proteins in inclusion bodies is a hallmark of many neurodegenerative diseases. Impairment of proteolytic systems is a common event in these protein misfolding diseases. Recently, mutations in the UBQLN 2 gene encoding ubiquilin 2 have been identified in X-linked amyotrophic lateral sclerosis (ALS). Furthermore, ubiquilin 2 is associated with inclusions in familial and sporadic ALS/dementia, synucleinopathies and polyglutamine diseases. Ubiquilin 2 exerts a regulatory role in proteostasis and thus it has been suggested that ubiquilin 2 pathology may be a common event in neurodegenerative diseases. Tauopathies, a heterogenous group of neurodegenerative diseases accompanied with dementia, are characterized by inclusions of the microtubule-binding protein tau. In the present study, we investigate whether ubiquilin 2 is connected with tau pathology in Alzheimer's disease (AD), supranuclear palsy (PSP) and Pick's disease (PiD) and familial cases with frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We show that ubiquilin 2 positive inclusions are absent in these tauopathies. Furthermore, we find decreased ubiquilin 2 protein levels in AD patients, but our results do not indicate a correlation with tau pathology. Our data show no evidence for involvement of ubiquilin 2 and indicate that other mechanisms underly the proteostatic disturbances in tauopathies.

Immunohistochemical characterization of novel monoclonal antibodies against the N-terminus of amyloid ß-peptide.

Abstract Amyloid ß-peptide (Aß) is a key molecule in Alzheimer's disease (AD). Reliable immunohistochemical (IHC) methods to detect Aß and Aß-associated factors (AAF) in brain specimens are needed to determine their role in AD pathophysiology. Formic acid (FA) pre-treatment, which is generally used to enable efficient detection of Aß with IHC, induces structural modifications within the Aß, as well as in AAF. Consequently, interpretation of double IHC stainings becomes difficult. Therefore, serial stainings of two newly produced monoclonal antibodies (mAbs) VU-17 and IC16 and two other mAbs (6E10 and 3D6) were performed with four different pre-treatments (no pre-treatment, Tris/EDTA, citrate and FA) and additionally six IHC characteristics were scored: diffuse/compact/classic plaques, arteries with cerebral Aß angiopathy, dyshoric angiopathy, capillaries with dyshoric angiopathy. Subsequently, these stainings were compared with IHC procedures, which are frequently used in a diagnostic setting, employing mAbs 4G8 and 6F/3D with FA pre-treatment. IHC Aß patterns obtained with VU-17 and, IC16 and 3D6 without the use of FA pre-treatment were comparable to those obtained with 4G8 and 6F/3D upon FA pre-treatment. Omission of FA pre-treatment gives the advantage to allow double IHC stainings, detecting both Aß and AAF that otherwise would have been structural modificated upon FA pre-treatment.

Unfolded protein response activates glycogen synthase kinase-3 via selective lysosomal degradation.

The unfolded protein response (UPR) is a stress response that is activated upon disturbed homeostasis in the endoplasmic reticulum. In Alzheimer's disease, as well as in other tauopathies, the UPR is activated in neurons that contain early tau pathology. A recent genome-wide association study identified genetic variation in a UPR transducer as a risk factor for tauopathy, supporting a functional connection between UPR activation and tau pathology. Here we show that UPR activation increases the activity of the major tau kinase glycogen synthase kinase (GSK)-3 in vitro via a selective removal of inactive GSK-3 phosphorylated at Ser(21/9). We demonstrate that this is mediated by the autophagy/lysosomal pathway. In brain tissue from patients with different tauopathies, lysosomal accumulations of pSer(21/9) GSK-3 are found in neurons with markers for UPR activation. Our data indicate that UPR activation increases the activity of GSK-3 by a novel mechanism, the lysosomal degradation of the inactive pSer(21/9) GSK-3. This may provide a functional explanation for the close association between UPR activation and early tau pathology in neurodegenerative diseases.

Intracellular accumulation of aggregated pyroglutamate amyloid beta: convergence of aging and Aß pathology at the lysosome.

Deposition of aggregated amyloid beta (Aß) is a major hallmark of Alzheimer's disease (AD)-a common age-related neurodegenerative disorder. Typically, Aß is generated as a peptide of varying lengths. However, a major fraction of Aß peptides in the brains of AD patients has undergone posttranslational modifications, which often radically change the properties of the peptides. Aß3(pE)-42 is an N-truncated, pyroglutamate-modified variant that is abundantly present in AD brain and was suggested to play a role early in the pathogenesis. Here we show that intracellular accumulation of oligomeric aggregates of Aß3(pE)-42 results in loss of lysosomal integrity. Using a novel antibody specific for aggregates of AßpE3, we show that in postmortem human brain tissue, aggregated AßpE3 is predominantly found in the lysosomes of both neurons and glial cells. Our data further demonstrate that AßpE3 is relatively resistant to lysosomal degradation, which may explain its accumulation in the lysosomes. The intracellular AßpE3 aggregates increase in an age-dependent manner. The results presented in this study support a model where Aß pathology and aging converge, leading to accumulation of the degradation-resistant pE-modified Aß in the lysosomes, lysosomal dysfunction, and neurodegeneration.

Disturbed Ca2+ homeostasis increases glutaminyl cyclase expression; connecting two early pathogenic events in Alzheimer's disease in vitro.

A major neuropathological hallmark of Alzheimer's disease (AD) is the deposition of aggregated ß amyloid (Aß) peptide in the senile plaques. Aß is a peptide of 38-43 amino acids and its accumulation and aggregation plays a key role early in the disease. A large fraction of ß amyloid is N-terminally truncated rendering a glutamine that can subsequently be cyclized into pyroglutamate (pE). This makes the peptide more resistant to proteases, more prone to aggregation and increases its neurotoxicity. The enzyme glutaminyl cyclase (QC) catalyzes this conversion of glutamine to pE. In brains of AD patients, the expression of QC is increased in the earliest stages of pathology, which may be an important event in the pathogenesis. In this study we aimed to investigate the regulatory mechanism underlying the upregulation of QC expression in AD. Using differentiated SK-N-SH as a neuronal cell model, we found that neither the presence of Aß peptides nor the unfolded protein response, two early events in AD, leads to increased QC levels. In contrast, we demonstrated increased QC mRNA levels and enzyme activity in response to another pathogenic factor in AD, perturbed intracellular Ca(2+) homeostasis. The QC promoter contains a putative binding site for the Ca(2+) dependent transcription factors c-fos and c-jun. C-fos and c-jun are induced by the same Ca(2+)-related stimuli as QC and their upregulation precedes QC expression. We show that in the human brain QC is predominantly expressed by neurons. Interestingly, the Ca(2+)- dependent regulation of both c-fos and QC is not observed in non-neuronal cells. Our results indicate that perturbed Ca(2+) homeostasis results in upregulation of QC selectively in neuronal cells via Ca(2+)- dependent transcription factors. This suggests that disruption of Ca(2+) homeostasis may contribute to the formation of the neurotoxic pE Aß peptides in Alzheimer's disease.

Neuroinflammation and common mechanism in Alzheimer's disease and prion amyloidosis: amyloid-associated proteins, neuroinflammation and neurofibrillary degeneration.

BACKGROUND: In cases with a long (>1 year) clinical duration of prion disease, the prion protein can form amyloid deposits. These cases do not show accumulation of 4-kDa ß-amyloid, which is observed in amyloid deposits in Alzheimer's disease (AD). In AD, amyloid is associated with inflammation and neurofibrillary degeneration, and it is elusive whether prion amyloid is associated with these changes as well. OBJECTIVES: The presence of inflammation and neurofibrillary degeneration was evaluated in prion amyloidosis. MATERIAL AND METHODS: Cortical areas of variant Creutzfeldt-Jakob disease (CJD; n = 3), young sporadic CJD (n = 4), different Gerstmann-Sträussler-Scheinker's disease patients (n = 5) and AD cases (n = 5) were examined using immunohistochemistry and specific stainings for amyloid. RESULTS: In both AD and prion disease cases, which were negative for 4-kDa ß-amyloid, parenchymal and vascular amyloid deposits were positive for amyloid-associated proteins such as complement protein and were associated with microglia clusters. Tau and ubiquitin were found near prion plaques in some of the Gerstmann-Sträussler-Scheinker's disease and sporadic CJD cases and also near vascular prion amyloid deposits. In variant CJD cases, occasionally, microglia clustering was found in plaques but no ubiquitin or complement proteins and hardly tau protein. CONCLUSIONS: In both AD and prion disease amyloid formation, irrespective of the protein involved, there seems to be a neuroinflammatory response with secondary neurofibrillary degeneration.

Activation of the unfolded protein response is an early event in Alzheimer's and Parkinson's disease.

BACKGROUND: Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by the accumulation and aggregation of misfolded proteins. Disturbed homeostasis in the endoplasmic reticulum leads to accumulation of misfolded proteins, which triggers a stress response called the unfolded protein response (UPR) that protects the cell against the toxic buildup of misfolded proteins. OBJECTIVE: In this paper, we will briefly review the early involvement of the UPR in the pathology of AD and PD. METHODS: Expression of UPR activation markers was analyzed in human brain tissue using immunohistochemistry and Western blot analysis. RESULTS: Neuropathological studies demonstrate that UPR activation markers are increased in neurons in AD and PD. In AD, UPR activation markers are observed in neurons with diffuse staining of phosphorylated tau protein. In PD, increased immunoreactivity for UPR activation markers is detected in neuromelanin containing dopaminergic neurons of the substantia nigra, which colocalize with diffuse α-synuclein staining. CONCLUSION: UPR activation is closely associated with the first stages of accumulation and aggregation of the toxic proteins involved in AD and PD. Studies of postmortem brain tissue indicate that UPR activation is an early event in neurodegeneration.

The unfolded protein response is associated with early tau pathology in the hippocampus of tauopathies.

The unfolded protein response (UPR) is a stress response activated upon disturbed homeostasis in the endoplasmic reticulum (ER). Previously, we reported that the activation of the UPR closely correlates with the presence of phosphorylated tau (p-tau) in Alzheimer's disease (AD). As well as increased presence of intracellular p-tau, AD brains are characterized by extracellular deposits of ß amyloid (Aß). Recent in vitro studies have shown that Aß can induce ER stress and activation of the UPR. The aim of the present study is to investigate UPR activation in sporadic tauopathies like progressive supranuclear palsy (PSP) and Pick's disease (PiD), and familial cases with frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) which carry mutations in the gene encoding for tau (MAPT). The presence of phosphorylated pancreatic ER kinase (pPERK) and phosphorylated inositol requiring enzyme 1α (pIRE1), which are indicative of an activated UPR, was assessed by immunohistochemistry in cases neuropathologically defined as frontotemporal lobar degeneration with tau pathology (FTLD-tau). Increased presence of UPR activation markers pPERK and pIRE1 was observed in neurons and glia in FTLD-tau cases, in contrast to FTLD subtypes negative for tau pathology or in non-neurological controls. pPERK and pIRE1 were also prominently present in relatively young carriers of MAPT mutation. A strong association between the presence of UPR activation markers and p-tau was observed in the hippocampus of FTLD-tau cases. Double immunohistochemical staining on FTLD-tau cases revealed that UPR activation is predominantly observed in neurons that show diffuse staining of p-tau. These data demonstrate that UPR activation is intimately connected with the accumulation and aggregation of p-tau, and occurs independently from Aß deposits. Our findings provide new pathological insight into the close association between p-tau and UPR activation in tauopathies.

Neuroinflammation in Alzheimer's disease wanes with age.

BACKGROUND: Inflammation is a prominent feature in Alzheimer's disease (AD). It has been proposed that aging has an effect on the function of inflammation in the brain, thereby contributing to the development of age-related diseases like AD. However, the age-dependent relationship between inflammation and clinical phenotype of AD has never been investigated. METHODS: In this study we have analysed features of the neuroinflammatory response in clinically and pathologically confirmed AD and control cases in relation to age (range 52-97 years). The mid-temporal cortex of 19 controls and 19 AD cases was assessed for the occurrence of microglia and astrocytes by immunohistochemistry using antibodies directed against CD68 (KP1), HLA class II (CR3/43) and glial fibrillary acidic protein (GFAP). RESULTS: By measuring the area density of immunoreactivity we found significantly more microglia and astrocytes in AD cases younger than 80 years compared to older AD patients. In addition, the presence of KP1, CR3/43 and GFAP decreases significantly with increasing age in AD. CONCLUSION: Our data suggest that the association between neuroinflammation and AD is stronger in relatively young patients than in the oldest patients. This age-dependent relationship between inflammation and clinical phenotype of AD has implications for the interpretation of biomarkers and treatment of the disease.

Apolipoprotein E and LRP1 Increase Early in Parkinson's Disease Pathogenesis.

Parkinson's disease (PD) is characterized by α-synuclein-containing Lewy bodies (LBs) and loss of melanized neurons in the substantia nigra (SN). Recently, a link between apolipoprotein E (ApoE) expression, α-synuclein aggregation, and neurodegeneration was suggested. Here, we report on ApoE expression appearing in melanized neurons of the SN and in LBs in both PD and incidental LB disease cases. Interestingly, increased expression of the low-density lipoprotein receptor-related protein 1 (the receptor for ApoE) was also observed in incidental LB disease and PD. Our data suggest that alterations in lipoprotein homeostasis/signaling in melanized neurons of the SN are an early event during PD pathogenesis.

Characteristics of dyshoric capillary cerebral amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) affects brain parenchymal and leptomeningeal arteries and arterioles but sometimes involves capillaries (capCAA) with spread of the amyloid into the surrounding neuropil, that is, dyshoric changes. We determined the relationship between capCAA and larger vessel CAA, ß amyloid (Aß) plaques, neurofibrillary changes, inflammation, and apolipoprotein E (APOE) in 22 cases of dyshoric capCAA using immunohistochemistry. The dyshoric changes contained predominantly Aß1-40, whereas dense bulblike deposits adjacent to the capillary wall contained mostly Aß1-42. There was an inverse local correlation between Aß plaque load and capCAA severity (p = 0.01), suggesting that Aß transport between the neuropil and the circulation may be mechanistically involved. Deposits of hyperphosphorylated tau and ubiquitin and clusters of activated microglia, resembling the changes around Aß plaques, were found around capCAA but were absent around larger vessel CAA. In 14 cases for which APOE genotype was available, there was a high APOE-ε4 allele frequency (54%; 43% homozygous). The severity of CapCAA increased with the number of ε4-alleles; and APOE4 seemed to colocalize with capCAA by immunohistochemistry. These results suggest that capCAA is pathologically and possibly pathogenetically distinct from larger vessel CAA, and that it is associated with a high APOE-ε4 allele frequency.

Morphometric changes in the cortical microvascular network in Alzheimer's disease.

Alzheimer's disease (AD) pathology is accompanied by abnormalities of the microvasculature. Despite the potential importance of morphometric changes in the cortical capillary network on neuronal dysfunction and cognitive impairment, few autopsy studies have addressed this issue. In the present study, we investigated morphological microvascular changes and capillary length density (CLD) in ten well-characterized AD patients compared to ten age-matched controls using virtual isotropic hemispheres. The CLD in the temporal cortex was increased by 33% in AD patients compared to controls (p=0.04), whereas CLD in the occipital cortex was unchanged. An increase of CLD was correlated to a decrease of cortical diameter in the temporal cortex (Pearson's r -0.62, p=0.003), suggesting that the increase in temporal CLD results from, or contributes to cortical atrophy. In the occipital cortex, more string vessels, probably remnants of degenerated capillaries, were observed in AD patients than in controls (p=0.004). An exploratory analysis suggests co-localization of Aß and string vessels. Our data indicate that morphometric changes in the cortical capillary network occur in AD in a region-specific manner and may be related to cortical atrophy in the affected regions.

The unfolded protein response is activated in pretangle neurons in Alzheimer's disease hippocampus.

Accumulation of misfolded proteins in the endoplasmic reticulum triggers a cellular stress response called the unfolded protein response (UPR) that protects the cell against the toxic buildup of misfolded proteins. Previously, we reported that UPR activation is increased in Alzheimer's disease (AD) patients. How the UPR relates to the pathological hallmarks of AD is still elusive. In the present study, the involvement of UPR activation in neurofibrillary degeneration in AD was investigated. Immunoreactivity for the phosphorylated UPR activation markers pancreatic ER kinase (pPERK), eukaryotic initiation factor 2alpha, and inositol-requiring enzyme 1alpha was observed in hippocampal neurons associated with granulovacuolar degeneration. The percentage of pPERK-immunoreactive neurons was increased in AD cases compared with nondemented control cases and with the Braak stage for neurofibrillary changes. Although absent from neurofibrillary tangles, pPERK immunoreactivity was most abundant in neurons with diffuse localization of phosphorylated tau protein. Additional analyses showed that pPERK immunoreactivity was associated with ubiquitin and the ubiquitin binding protein p62. A strong co-occurrence of immunoreactivity for both pPERK and glycogen synthase kinase 3beta in neurons was also observed. Together, these data indicate that UPR activation in AD neurons occurs at an early stage of neurofibrillary degeneration and suggest that the prolonged activation of the UPR is involved in both tau phosphorylation and neurodegeneration in AD pathogenesis.

Increased expression of the oligopeptidase THOP1 is a neuroprotective response to Abeta toxicity.

In a comprehensive proteomics study aiming at the identification of proteins associated with amyloid-beta (Abeta)-mediated toxicity in cultured cortical neurons, we have identified Thimet oligopeptidase (THOP1). Functional modulation of THOP1 levels in primary cortical neurons demonstrated that its overexpression was neuroprotective against Abeta toxicity, while RNAi knockdown made neurons more vulnerable to amyloid peptide. In the TgCRND8 transgenic mouse model of amyloid plaque deposition, an age-dependent increase of THOP1 expression was found in brain tissue, where it co-localized with Abeta plaques. In accordance with these findings, THOP1 expression was significantly increased in human AD brain tissue as compared to non-demented controls. These results provide compelling evidence for a neuroprotective role of THOP1 against toxic effects of Abeta in the early stages of AD pathology, and suggest that the observed increase in THOP1 expression might be part of a compensatory defense mechanism of the brain against an increased Abeta load.

CC chemokine receptor 5 gene promoter activation by the cyclic AMP response element binding transcription factor.

The chemokine receptor CCR5 is implicated in the pathogenesis of various inflammatory diseases, such as multiple sclerosis (MS), atherosclerosis, transplant rejection, and autoimmunity. In previous studies, we have shown that MS lesions are characterized by enhanced expression of transcription factors associated with stress responses, ie, IRF-1, NF-kappaB, and CREB-1, which modulate expression of both classes of major histocompatibility complex (MHC) molecules. The expression of MHC-I and MHC-II molecules greatly overlaps with the expression of CCR5 in MS lesions. Therefore, we investigated whether these factors are also involved in the transcriptional regulation of CCR5. Using in vitro assays, we determined that neither IRF-1 nor NF-kappaB is involved in the activation of the CCR5 promoter. This is corroborated by the finding that these factors are not involved in the induction of endogenous CCR5 transcription in various cell types. In contrast, we show that CCR5 expression is regulated by the cAMP/CREB pathway and that interference in this pathway affects endogenous CCR5 transcription. From this, we conclude that the cAMP/CREB pathway is involved in the regulation of CCR5 transcription and that, given the ubiquitous nature of CREB-1 protein expression, additional regulatory mechanisms must contribute to cell type-specific expression of CCR5.

Microglia activation in sepsis: a case-control study.

BACKGROUND: Infection induces an acute phase response that is accompanied by non-specific symptoms collectively named sickness behavior. Recent observations suggest that microglial cells play a role in mediating behavioral changes in systemic infections. In animal models for sepsis it has been shown that after inducing lipopolysaccharide, LPS, microglia in the brain were activated. The aim of this study was to investigate whether activation of microglia can be detected in patients who died of sepsis. METHODS: In a case-control study brain tissue of 13 patients who died with sepsis was compared with that of 17 controls. Activated microglia were identified by expression of MHC-class II antigens and CD68. Microglia activation was analyzed by a semiquantitative score combining both the number of the immunoreactive cells and their morphology. RESULTS: In patients who died with sepsis there was a significant increase in activated microglia in the grey matter when stained with CD68 compared to controls. This effect was independent of the effect of age. CONCLUSION: This study shows for the first time in human brain tissue an association between a systemic infection and activation of microglia in the brain. Activated microglia during sepsis could play a role in behavioral changes associated with systemic infection.

DNA polymerase-beta is expressed early in neurons of Alzheimer's disease brain and is loaded into DNA replication forks in neurons challenged with beta-amyloid.

Cultured neurons exposed to synthetic beta-amyloid (Abeta) fragments reenter the cell cycle and initiate a pathway of DNA replication that involves the repair enzyme DNA polymerase-beta (DNA pol-beta) before undergoing apoptotic death. In this study, by performing coimmunoprecipitation experiments on cross-linked nucleoprotein fragments from Abeta-treated neurons, we demonstrate that DNA pol-beta coimmunoprecipitates with cell division cycle 45 (Cdc45) and with DNA primase in short nucleoprotein fragments. This indicates that DNA pol-beta is loaded into neuronal DNA replication forks after Abeta treatment. In response to Abeta the canonical DNA-synthesizing enzyme DNA pol-delta also was loaded into neuronal replication forks, but at later times than DNA pol-beta. Methoxyamine, an inhibitor of the apurinic/apyrimidinic endonuclease that allows for the recruitment of DNA pol-beta during the process of base excision repair (BER), failed to affect coimmunoprecipitation between DNA pol-beta and Cdc45, indicating that DNA pol-beta loading to the replication forks is independent of DNA breaks. However, methoxyamine reduced DNA replication and ensuing apoptosis in neurons exposed to Abeta, suggesting that an efficient BER process allows DNA replication to proceed up to the threshold for death. These data demonstrate that DNA pol-beta is an essential component of the DNA replication machinery in Abeta-treated neurons and additionally support the hypothesis of a close association of cell cycle events with neuronal death in Alzheimer's disease (AD). Accordingly, by investigating the neuronal expression of DNA pol-beta, along with phosphorylated retinoblastoma protein and neurofibrillary changes in AD brain, we show an early involvement of DNA pol-beta in the pathogenesis of AD.