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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., (Copyright © 2016 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.)

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-1β 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-1β 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.

The unfolded protein response (UPR) is activated in neurodegenerative tauopathies such as Alzheimer's disease (AD) in close connection with early stages of tau pathology. Metabolic disturbances are strongly associated with increased risk for AD and are a potent inducer of the UPR. Here, we demonstrate that metabolic stress induces the phosphorylation of endogenous tau via activation of the UPR. Strikingly, upon restoration of the metabolic homeostasis, not only the levels of the UPR markers pPERK, pIRE1α and BiP, but also tau phosphorylation are reversed both in cell models as well as in torpor, a physiological hypometabolic model in vivo. Intervention in the UPR using the global UPR inhibitor TUDCA or a specific small-molecule inhibitor of the PERK signaling pathway, inhibits the metabolic stress-induced phosphorylation of tau. These data support a role for UPR-mediated tau phosphorylation as part of an adaptive response to metabolic stress. Failure to restore the metabolic homeostasis will lead to prolonged UPR activation and tau phosphorylation, and may thus contribute to AD pathogenesis. We demonstrate that the UPR is functionally involved in the early stages of tau pathology. Our data indicate that targeting of the UPR may be employed for early intervention in tau-related neurodegenerative diseases.

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.

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.

Abstract Amyloid β-peptide (Aβ) is a key molecule in Alzheimer's disease (AD). Reliable immunohistochemical (IHC) methods to detect Aβ and Aβ-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 Aβ with IHC, induces structural modifications within the Aβ, 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 Aβ 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 Aβ 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 Aβ and AAF that otherwise would have been structural modificated upon FA pre-treatment.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Deposition of aggregated amyloid beta (Aβ) is a major hallmark of Alzheimer's disease (AD)-a common age-related neurodegenerative disorder. Typically, Aβ is generated as a peptide of varying lengths. However, a major fraction of Aβ peptides in the brains of AD patients has undergone posttranslational modifications, which often radically change the properties of the peptides. Aβ3(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 Aβ3(pE)-42 results in loss of lysosomal integrity. Using a novel antibody specific for aggregates of AβpE3, we show that in postmortem human brain tissue, aggregated AβpE3 is predominantly found in the lysosomes of both neurons and glial cells. Our data further demonstrate that AβpE3 is relatively resistant to lysosomal degradation, which may explain its accumulation in the lysosomes. The intracellular AβpE3 aggregates increase in an age-dependent manner. The results presented in this study support a model where Aβ pathology and aging converge, leading to accumulation of the degradation-resistant pE-modified Aβ in the lysosomes, lysosomal dysfunction, and neurodegeneration.

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 β amyloid (Aβ). Recent in vitro studies have shown that Aβ 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 Aβ deposits. Our findings provide new pathological insight into the close association between p-tau and UPR activation in tauopathies., (Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

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., (Copyright © 2012 S. Karger AG, Basel.)

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 β-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 β-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., (Copyright © 2012 S. Karger AG, Basel.)

A major neuropathological hallmark of Alzheimer's disease (AD) is the deposition of aggregated β amyloid (Aβ) peptide in the senile plaques. Aβ 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 β 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 Aβ 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 Aβ peptides in Alzheimer's disease.

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., (© 2011 Hoozemans et al; licensee BioMed Central Ltd.)

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., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Protein folding stress in the endoplasmic reticulum (ER) may lead to activation of the unfolded protein response (UPR), aimed to restore cellular homeostasis via transcriptional and post-transcriptional mechanisms. ER stress is also reported to activate the ER overload response (EOR), which activates transcription via NF-κB. We previously demonstrated that UPR activation is an early event in pre-tangle neurons in Alzheimer's disease (AD) brain. Misfolded and unfolded proteins are degraded via the ubiquitin proteasome system (UPS) or autophagy. UPR activation is found in AD neurons displaying both early UPS pathology and autophagic pathology. Here we investigate whether activation of the UPR and/or EOR is employed to enhance the proteolytic capacity of neuronal cells. Expression of the immunoproteasome subunits β2i and β5i is increased in AD brain. However, expression of the proteasome subunits is not increased by the UPR or EOR. UPR activation does not relocalize the proteasome or increase overall proteasome activity. Therefore proteasomal degradation is not increased by ER stress. In contrast, UPR activation enhances autophagy and LC3 levels are increased in neurons displaying UPR activation in AD brain. Our data suggest that autophagy is the major degradational pathway following UPR activation in neuronal cells and indicate a connection between UPR activation and autophagic pathology in AD brain.

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, β amyloid (Aβ) plaques, neurofibrillary changes, inflammation, and apolipoprotein E (APOE) in 22 cases of dyshoric capCAA using immunohistochemistry. The dyshoric changes contained predominantly Aβ1-40, whereas dense bulblike deposits adjacent to the capillary wall contained mostly Aβ1-42. There was an inverse local correlation between Aβ plaque load and capCAA severity (p = 0.01), suggesting that Aβ 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 Aβ 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.

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 Aβ 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.

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.

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.

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.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the deposition of beta amyloid (Abeta) protein and the formation of neurofibrillary tangles. In addition, there is an increase of inflammatory proteins in the brains of AD patients. Epidemiological studies, indicating that non-steroidal anti-inflammatory drugs (NSAIDs) decrease the risk of developing AD, have encouraged the study on the role of inflammation in AD. The best-characterized action of most NSAIDs is the inhibition of cyclooxygenase (COX). The expression of the constitutively expressed COX-1 and the inflammatory induced COX-2 has been intensively investigated in AD brain and different disease models for AD. Despite these studies, clinical trials with NSAIDs or selective COX-2 inhibitors showed little or no effect on clinical progression of AD. The expression levels of COX-1 and COX-2 change in the different stages of AD pathology. In an early stage, when low-fibrillar Abeta deposits are present and only very few neurofibrillary tangles are observed in the cortical areas, COX-2 is increased in neurons. The increased neuronal COX-2 expression parallels and colocalizes with the expression of cell cycle proteins. COX-1 is primarily expressed in microglia, which are associated with fibrillar Abeta deposits. This suggests that in AD brain COX-1 and COX-2 are involved in inflammatory and regenerating pathways respectively. In this review we will discuss the role of COX-1 and COX-2 in the different stages of AD pathology. Understanding the physiological and pathological role of cyclooxygenase in AD pathology may facilitate the design of therapeutics for the treatment or prevention of AD.

Parkinson's disease (PD) is, at the neuropathological level, characterized by the accumulation of misfolded proteins. The presence of misfolded proteins can trigger a cellular stress response in the endoplasmic reticulum (ER) called the Unfolded Protein Response (UPR). The UPR has been shown to be involved in cellular models for PD. In this study, we investigated UPR activation in the substantia nigra of control and PD patients. Immunoreactivity for the UPR activation markers phosphorylated pancreatic ER kinase (pPERK) and phosphorylated eukaryotic initiation factor 2alpha (peIF2alpha) is detected in neuromelanin containing dopaminergic neurons in the substantia nigra of PD cases but not in control cases. In addition, pPERK immunoreactivity is colocalized with increased alpha-synuclein immunoreactivity in dopaminergic neurons. These data show that the UPR is activated in PD and that UPR activation is closely associated with the accumulation and aggregation of alpha-synuclein.

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.

Matrix metalloproteinases (MMPs) are proteases known for their capacity to degrade extracellular matrix (ECM) components. MMPs have been implicated in several central nervous system (CNS) diseases, including multiple sclerosis (MS). Microarray analysis has demonstrated significant increased mRNA levels of MMP-19 in chronic MS lesions, suggesting a role of MMP-19 in MS pathogenesis. Therefore, in this study, we investigated the expression pattern and cellular localization of MMP-19 protein in various well-characterized MS lesion stages. In normal control patient white matter, MMP-19 was constitutively expressed by microglia throughout the brain parenchyma, suggesting a physiological role for this MMP family member. Likewise, MMP-19 was expressed by microglia in (p)reactive MS lesions, albeit more intense. In highly active demyelinating MS lesions, parenchymal and perivascular myelin-laden macrophages were strongly immunoreactive for MMP-19, whereas reactive astrocytes were occasionally immunopositive. Astrocytes in chronic inactive lesions were weakly stained for MMP-19. In vitro, MMP-19 was expressed in cultures of primary human microglia, not in astrocyte cultures. As MMP-19 is able to degrade basement membrane constituents and other ECM proteins, it is conceivable that this relatively novel MMP family member contributes to MS pathology by remodelling the ECM of the CNS, thereby influencing leucocyte infiltration, axonal regeneration and astrogliosis.

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.

Alzheimer's disease (AD) is characterized by the accumulation and aggregation of misfolded proteins. The presence of misfolded proteins in the endoplasmic reticulum (ER) triggers a cellular stress response called the unfolded protein response (UPR). Previously, we have shown that the UPR is activated in AD neurons. In actively dividing cells, activation of the UPR is accompanied by decreased cell cycle protein expression and an arrest in the G1 phase of the cell cycle. Aberrant expression of cell cycle proteins has been observed in post mitotic neurons in AD and is suggested to be involved in neurodegeneration. In this study we show that the protein levels of BiP/GRP78, an ER-stress marker, is increased in Braak stages B and C for amyloid deposits. This is in contrast to the levels of cell cycle markers cyclin D1, cyclin E and phosphorylated retinoblastoma protein (ppRb) which are decreased in Braak stage C compared to Braak stage A for amyloid deposits. In addition, we report a negative correlation between neuronal expression of ppRb and expression levels of BiP/GRP78 in control and AD cases. Activation of the UPR in neuronal cells induces changes in cell cycle protein expression similar to these observed in AD brain. ER stress inducers tunicamycin and thapsigargin down-regulate cell cycle proteins ppRb and cyclin D1 in differentiated neuroblastoma cells. In contrast, protein levels of p27, a cyclin dependent kinase inhibitor, are increased after induction of ER-stress using tunicamycin. These data suggest that activation of the UPR affects cell cycle protein expression in neurons during neurodegeneration in AD.

Statin treatment is proposed to be a new potential therapy for multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system. The effects of statin treatment on brain cells, however, are hardly understood. We therefore evaluated the effects of simvastatin treatment on the migratory capacity of brain microglial cells, key elements in the pathogenesis of MS. It is shown that exposure of human and murine microglial cells to simvastatin reduced cell surface expression of the chemokine receptors CCR5 and CXCR3. In addition, simvastatin treatment specifically abolished chemokine-induced microglial cell motility, altered actin cytoskeleton distribution, and led to changes in intracellular vesicles. These data clearly show that simvastatin inhibits several immunological properties of microglia, which may provide a rationale for statin treatment in MS., (2005 Wiley-Liss, Inc.)

Postmortem MRI-guided tissue sampling significantly enhances the yield of MS lesions in autopsy material, but so far it is unknown whether abnormalities concur with blood-brain barrier alterations. Here we sampled MS lesions with focal and diffuse abnormalities (diffusely abnormal white matter; DAWM) on MRI; both were coupled to the presence of MS lesions upon neuropathological examination. Extravascular distribution of fibrinogen, indicating BBB disturbance, was observed in so-called (p)reactive lesions that reflect discrete areas of microglial activation without demyelination within an otherwise normal appearing white matter. Leakage became more extensive in active demyelinating MS lesions to chronic inactive lesions. An enlargement of the perivascular (Virchow-Robin) space containing infiltrated leukocytes was associated with both DAWM and focal abnormalities on postmortem MRI. This study shows for the first time that in MS brain changes in the vasculature take place not only in focal lesions but also in DAWM as detected by postmortem MRI.

Neuronal expression of cyclooxygenase-2 (COX-2) and cell cycle proteins is suggested to contribute to neurodegeneration during Alzheimer's disease (AD). The stimulus that induces COX-2 and cell cycle protein expression in AD is still elusive. Activated glia cells are shown to secrete substances that can induce expression of COX-2 and cell cycle proteins in vitro. Using post mortem brain tissue we have investigated whether activation of microglia and astrocytes in AD brain can be correlated with the expression of COX-2 and phosphorylated retinoblastoma protein (ppRb). The highest levels of neuronal COX-2 and ppRb immunoreactivity are observed in the first stages of AD pathology (Braak 0-II, Braak A). No significant difference in COX-2 or ppRb neuronal immunoreactivity is observed between Braak stage 0 and later Braak stages for neurofibrillary changes or amyloid plaques. The mean number of COX-2 or ppRb immunoreactive neurons is significantly decreased in Braak stage C compared to Braak stage A for amyloid deposits. Immunoreactivity for glial markers KP1, CR3/43 and GFAP appears in the later Braak stages and is significantly increased in Braak stage V-VI compared to Braak stage 0 for neurofibrillary changes. In addition, a significant negative correlation is observed between the presence of KP1, CR3/43 and GFAP immunoreactivity and the presence of neuronal immunoreactivity for COX-2 and ppRb. These data show that maximal COX-2 and ppRb immunoreactivity in neurons occurs during early Braak stages prior to the maximal activation of astrocytes and microglia. In contrast to in vitro studies, post mortem data do not support a causal relation between the activation of microglia and astrocytes and the expression of neuronal COX-2 and ppRb in the pathological cascade of AD.

Perivascular macrophages (PVM) constitute a subpopulation of resident macrophages in the central nervous system (CNS) that by virtue of their strategic location at the blood-brain barrier potentially lend themselves to a variety of important functions in both health and disease. Functional evidence suggests that PVM play a supportive role during experimental autoimmune encephalomyelitis in rodents. However, the function of PVM in the human CNS remains poorly characterized. We first set out to investigate the validity of the antibody EDhu1, which recognizes human CD163, to specifically identify human PVM. Second, we wanted to gain insight into the function of PVM in antigen recognition and presentation and therefore we studied the expression of DC-SIGN, mannose receptor, MHC class II, and several costimulatory molecules by PVM in the normal and inflamed human CNS (multiple sclerosis (MS) brain lesions). Conventional immunohistochemistry and double-labeled immunofluorescence techniques were used. We show that CD163 specifically reveals PVM in the normal human CNS. In MS lesions, CD163 staining reveals expression on foamy macrophages and microglia, besides an upregulation of the amount of PVM stained. In contrast, mannose receptor expression is restricted to PVM in both normal and inflamed brain tissue. Furthermore, we show that a subpopulation of PVM in the human brain express several molecules involved in antigen recognition, presentation, and costimulation. Therefore PVM, which occupy a strategic location at the BBB, are equipped to recognize antigen and present it to T cells, supporting a role in the regulation of perivascular inflammation in the human CNS., ((c) 2005 Wiley-Liss, Inc.)

Alzheimer's disease (AD) is, at the neuropathological level, characterized by the accumulation and aggregation of misfolded proteins. The presence of misfolded proteins in the endoplasmic reticulum (ER) triggers a cellular stress response called the unfolded protein response (UPR) that may protect the cell against the toxic buildup of misfolded proteins. In this study we investigated the activation of the UPR in AD. Protein levels of BiP/GRP78, a molecular chaperone which is up-regulated during the UPR, was found to be increased in AD temporal cortex and hippocampus as determined by Western blot analysis. At the immunohistochemical level intensified staining of BiP/GRP78 was observed in AD, which did not co-localize with AT8-positive neurofibrillary tangles. In addition, we performed immunohistochemistry for phosphorylated (activated) pancreatic ER kinase (p-PERK), an ER kinase which is activated during the UPR. p-PERK was observed in neurons in AD patients, but not in non-demented control cases and did not co-localize with AT8-positive tangles. Overall, these data show that the UPR is activated in AD, and the increased occurrence of BiP/GRP78 and p-PERK in cytologically normal-appearing neurons suggest a role for the UPR early in AD neurodegeneration. Although the initial participation of the UPR in AD pathogenesis might be neuroprotective, sustained activation of the UPR in AD might initiate or mediate neurodegeneration.

An important role for CX3CL1 in neuroinflammation and neurodegeneration has been suggested in recent publications. In this study, we compared the expression of CX3CL1 and its receptor CX3CR1 in human brain tissue derived from control patients without neurological complications and in multiple sclerosis (MS) patients. Results from this study demonstrate that CX3CL1 is constitutively expressed in human central nervous system (CNS) astrocytes in vivo and under basal conditions in human adult astrocyte cultures. CX3CR1 is expressed on astrocytes and microglial cells both in vivo and in vitro. Chemotaxis assay shows a functional response upon CX3CR1 signaling in microglial cells. Although CX3CL1 expression is upregulated in cultured astrocytes in response to proinflammatory cytokines, no evidence for expression differences of CX3CL1 between control patients and MS patients was found. Our data suggest that CX3CL1 has more general physiological functions, which occur also in the absence of proinflammatory conditions.

Matrix metalloproteinases (MMPs) are proteases involved in extracellular matrix (ECM) remodeling, leukocyte infiltration into lesions and myelin degradation in the central nervous system (CNS) disease multiple sclerosis (MS). We have investigated whether MMP-12 (macrophage metalloelastase) is expressed in MS lesions at various stages. In control patient tissue and (p)reactive MS lesions, only occasional microglial and astrocyte staining was detected. In contrast, in active demyelinating lesions, phagocytic macrophages were MMP-12 positive. A lower proportion of phagocytes was positive for MMP-12 in chronic active demyelinating lesions and inactive lesions. This suggests a role for MMP-12 during demyelination in MS.