Biomedical subjects

Kathleen S Montine

Publications and source records attributed to Kathleen S Montine.

At least 19 recordsLinked to original sources

PubMed2007

Prostaglandin E2 receptor subtype 2 (EP2) regulates microglial activation and associated neurotoxicity induced by aggregated alpha-synuclein.

BACKGROUND: The pathogenesis of idiopathic Parkinson's disease (PD) remains elusive, although evidence has suggested that neuroinflammation characterized by activation of resident microglia in the brain may contribute significantly to neurodegeneration in PD. It has been demonstrated that aggregated alpha-synuclein potently activates microglia and causes neurotoxicity. However, the mechanisms by which aggregated alpha-synuclein activates microglia are not understood fully. METHODS: We investigated the role of prostaglandin E2 receptor subtype 2 (EP2) in alpha-synuclein aggregation-induced microglial activation using ex vivo, in vivo and in vitro experimental systems. RESULTS: Results demonstrated that ablation of EP2(EP2-/-) significantly enhanced microglia-mediated ex vivo clearance of alpha-synuclein aggregates (from mesocortex of Lewy body disease patients) while significantly attenuating neurotoxicity and extent of alpha-synuclein aggregation in mice treated with a parkinsonian toxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Furthermore, we report that reduced neurotoxicity by EP2-/- microglia could be attributed to suppressed translocation of a critical cytoplasmic subunit (p47-phox) of NADPH oxidase (PHOX) to the membranous compartment after exposure to aggregated alpha-synuclein. CONCLUSION: Thus, it appears that microglial EP2 plays a critical role in alpha-synuclein-mediated neurotoxicity.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2006

Effects of chemical chaperones on oxidative stress and detergent-insoluble species formation following conditional expression of amyloid precursor protein carboxy-terminal fragment.

Oxidative stress, protein misfolding, protein complex formation, and detergent insolubility are biochemical features of Alzheimer's disease (AD). We tested the cause-and-effect relationships among these using MC65 human neuroblastoma cells that exhibit toxicity upon conditional expression of carboxy-terminal fragments (CTFs) of the human amyloid precursor protein (APP). Treatments with three different antioxidants (alpha-tocopherol, N-acetyl cysteine, and alpha-lipoic acid) or three different compounds (glycerol, trimethylamine-N-oxide, and 4-phenylbutyric acid) that have been described to have a "chemical chaperone" function in promoting protein folding all had a protective effect on MC65 cells and decreased markers of oxidative damage and accumulation of high molecular weight amyloid (A) beta-immunoreactive (IR) species. However, chaperones partially reduced detergent insolubility of the remaining Abeta-IR species, while antioxidants did not. These results suggest that protein misfolding associated with overexpression of APP CTFs promotes oxidative stress and cytotoxicity and contributes to formation of detergent-insoluble species that appear unrelated to cytotoxicity.

Amyloid beta-Peptides↗

PubMed2006

Apolipoprotein E isoform-dependent dendritic recovery of hippocampal neurons following activation of innate immunity.

BACKGROUND: Innate immune activation, including a role for cluster of differentiation 14/toll-like receptor 4 co-receptors (CD14/TLR-4) co-receptors, has been implicated in paracrine damage to neurons in several neurodegenerative diseases that also display stratification of risk or clinical outcome with the common alleles of the apolipoprotein E gene (APOE): APOE2, APOE3, and APOE4. Previously, we have shown that specific stimulation of CD14/TLR-4 with lipopolysaccharide (LPS) leads to greatest innate immune response by primary microglial cultures from targeted replacement (TR) APOE4 mice and greatest p38MAPK-dependent paracrine damage to neurons in mixed primary cultures and hippocampal slice cultures derived from TR APOE4 mice. In contrast, TR APOE2 astrocytes had the highest NF-kappaB activity and no neurotoxicity. Here we tested the hypothesis that direct activation of CD14/TLR-4 in vivo would yield different amounts of paracrine damage to hippocampal sector CA1 pyramidal neurons in TR APOE mice. METHODS: We measured in vivo changes in dendrite length in hippocampal CA1 neurons using Golgi staining and determined hippocampal apoE levels by Western blot. Neurite outgrowth of cultured primary neurons in response to astrocyte conditioned medium was assessed by measuring neuron length and branch number. RESULTS: Our results showed that TR APOE4 mice had slightly but significantly shorter dendrites at 6 weeks of age. Following exposure to intracerebroventricular LPS, there was comparable loss of dendrite length at 24 hr among the three TR APOE mice. Recovery of dendrite length over the next 48 hr was greater in TR APOE2 than TR APOE3 mice, while TR APOE4 mice had failure of dendrite regeneration. Cell culture experiments indicated that the enhanced neurotrophic effect of TR APOE2 was LDL related protein-dependent. CONCLUSION: The data indicate that the environment within TR APOE2 mouse hippocampus was most supportive of dendrite regeneration while that within TR APOE4 hippocampus failed to support dendrite regeneration in this model of reversible paracrine damage to neurons from innate immune activation, and suggest an explanation for the stratification of clinical outcome with APOE seen in several degenerative diseases or brain that are associated with activated innate immune response.

Journal Article↗

PubMed2006

Apolipoprotein E-specific innate immune response in astrocytes from targeted replacement mice.

BACKGROUND: Inheritance of the three different alleles of the human apolipoprotein (apo) E gene (APOE) are associated with varying risk or clinical outcome from a variety of neurologic diseases. ApoE isoform-specific modulation of several pathogenic processes, in addition to amyloid beta metabolism in Alzheimer's disease, have been proposed: one of these is innate immune response by glia. Previously we have shown that primary microglia cultures from targeted replacement (TR) APOE mice have apoE isoform-dependent innate immune activation and paracrine damage to neurons that is greatest with TR by the epsilon4 allele (TR APOE4) and that derives from p38 mitogen-activated protein kinase (p38MAPK) activity. METHODS: Primary cultures of TR APOE2, TR APOE3 and TR APOE4 astrocytes were stimulated with lipopolysaccharide (LPS). ApoE secretion, cytokine production, and nuclear factor-kappa B (NF-kappaB) subunit activity were measured and compared. RESULTS: Here we showed that activation of primary astrocytes from TR APOE mice with LPS led to TR APOE-dependent differences in cytokine secretion that were greatest in TR APOE2 and that were associated with differences in NF-kappaB subunit activity. CONCLUSION: Our results suggest that LPS activation of innate immune response in TR APOE glia results in opposing outcomes from microglia and astrocytes as a result of TR APOE-dependent activation of p38MAPK or NF-kappaB signaling in these two cell types.

Journal Article↗

PubMed2006

Neurotoxicity from innate immune response is greatest with targeted replacement of E4 allele of apolipoprotein E gene and is mediated by microglial p38MAPK.

Inheritance of APOE alleles is associated with varying clinical outcomes in several neurodegenerative diseases that are associated with innate immune response in brain. We tested the hypothesis that inheritance of different APOE alleles would significantly modulate neurotoxicity arising from glial innate immune response. We first used dissociated cultures of wild-type (wt) murine neurons and glia derived from mice with targeted replacement (TR) of the epsilon2, epsilon3, or, epsilon4 APOE allele. Our results showed that the vast majority of bystander damage to wt neurons derived from microglia was greatest with TR APOE4 glia, intermediate from TR APOE3 glia, and least from TR APOE2 glia and preceded detectable NO secretion. Microglial p38MAPK-dependent cytokine secretion followed a similar pattern of TR APOE dependence. In hippocampal slice cultures, innate immune activation had a similar pattern of TR APOE-dependence and produced postsynaptic neuronal damage in TR APOE4 and TR APOE3 but not TR APOE2 cultures that was p38MAPK dependent. These findings suggest a new mechanism by which inheritance of different APOE alleles may influence the outcome of neurodegenerative diseases associated with microglial innate immune response.

Alleles↗

PubMed2006

Liquid chromatography with tandem mass spectrometry-based proteomic discovery in aging and Alzheimer's disease.

Systems biology offers enormous potential to understand the complexity of human brain aging and neurodegenerative diseases. Proteomics has an important role in these investigations because of its unique strengths and because of the potential central pathogenic contribution of pathological protein to several of these diseases. Here we have reviewed the methods and presented some examples of liquid chromatography-electrospray ionization-tandem mass spectrometry-based proteomics, with and without quantification using isotope-coded affinity tags, in the investigation of aging and Alzheimer's disease. As protocols and methods for improved quantitative high-throughput proteomics constantly improve, this approach will likely continue to provide deeper insight into human brain aging and neurodegenerative diseases.

Aging↗

PubMed2006

Selectively increased oxidative modifications mapped to detergent-insoluble forms of Abeta and beta-III tubulin in Alzheimer's disease.

Deleterious post-translational modifications (PTMs) to the neuronal cytoskeleton are a proposed mechanistic link between accumulation of amyloid (A) beta peptides and subsequent abnormalities of tau and neurodegeneration in Alzheimer's disease (AD). Here we tested the hypothesis that PTMs on neuronal tubulins selectively accumulate in a pathological protein fraction in AD. We used new software, P-MOD, to identify comprehensively and map PTMs using mass spectral data from soluble (normal) and detergent-insoluble (pathological) protein fractions from AD, as well as total extracts from controls, for selected proteins: Abeta, tau, apolipoprotein (apo) E, glial fibrillary acidic protein (GFAP), alpha-III tubulin, and beta-III tubulin. Our results confirmed direct observations of others by identifying methionine (M) sulfoxides at Abeta position 35 and numerous sites of tau phosphorylation in detergent-insoluble protein from AD, while no PTMs were enriched on primarily astrocyte-derived apoE or GFAP in this fraction. P-MOD mapped several abundant M sulfoxides to neuron-enriched beta-III tubulin but not its heterodimeric partner, neuron-enriched alpha-III tubulin, a result confirmed by selective suppression of CNBr-mediated cleavage of beta-III tubulin. These findings are the first comprehensive assessment of PTMs in AD and point to oxidative modification of beta-III tubulin as a potential contributor to the neuronal cytoskeletal disruption that is characteristic of AD.

Alzheimer Disease↗

PubMed2005

Proteomic determination of widespread detergent-insolubility including Abeta but not tau early in the pathogenesis of Alzheimer's disease.

Biochemical characterization of the major detergent-insoluble proteins that comprise hallmark histopathologic lesions initiated the molecular era of Alzheimer's disease (AD) research. Here, we reinvestigated detergent-insoluble proteins in AD using modern proteomic techniques. Using liquid chromatography (LC)-mass spectrometry (MS)-MS-based proteomics, we robustly identified 125 proteins in the detergent-insoluble fraction of late-onset AD (LOAD) temporal cortex that included several proteins critical to Abeta production, components of synaptic scaffolding, and products of genes linked to an increased risk of LOAD; we verified 15 of 15 of these proteins by Western blot. Following multiple analyses, we estimated that these represent ~80% of detergent-insoluble proteins in LOAD detectable by our method. Abeta, tau, and 7 of 8 other newly identified detergent-insoluble proteins were disproportionately increased in temporal cortex from patients with LOAD and AD derived from mutations in PSEN1 and PSEN2; all of these except tau were elevated in individuals with prodromal dementia, while none except Abeta were elevated in aged APPswe mice. These results are consistent with the amyloid hypothesis of AD and extend it to include widespread protein insolubility, not exclusively Abeta insolubility, early in AD pathogenesis even before the onset of clinical dementia.

Alzheimer Disease↗

PubMed2005

Suppression of murine cerebral F2-isoprostanes and F4-neuroprostanes from excitotoxicity and innate immune response in vivo by alpha- or gamma-tocopherol.

Oxidative damage to brain is a featured shared by several destructive and degenerative diseases and is thought to contribute to disease pathogenesis. Two commonly proposed sources of the increased free radical stress that leads to oxidative damage in several of these diseases are excitotoxicity and activation of innate immunity, both of which are proposed pharmacologic targets. Here we used models of excitotoxicity, intracerebroventricular (ICV) kainate (KA), and innate immune activation, ICV lipopolysaccharide (LPS), to test the effectiveness of peripherally administered alpha-tocopherol (AT) and gamma-tocopherol (GT) as neuroprotectants. We quantified murine cerebral oxidative damage by measuring F(2)-isoprostanes (IsoPs) and F(4)-neuroprostanes (NeuroPs) using stable isotope dilution methods followed by gas chromatography-mass spectrometry with selective ion monitoring. Our data showed that peripherally administered AT and GT were equally effective at suppressing acute oxidative damage from direct excitotoxicity caused by KA. In contrast, peripherally administered AT, but not GT, was effective at suppressing delayed neuronal oxidative damage from activated glial innate immune response. These data imply that AT may be more broadly protective of cerebrum from oxidative damage in different disease contexts.

Animals↗

PubMed2005

Proteomic analysis of neurofibrillary tangles in Alzheimer disease identifies GAPDH as a detergent-insoluble paired helical filament tau binding protein.

We performed proteomic analysis of neurofibrillary tangles (NFTs) obtained by laser capture microdissection from pyramidal neurons in hippocampal sector CA1 in patients with Alzheimer disease (AD) using liquid chromatography (LC)-mass spectrometry (MS)/MS. We discovered a total of 155 proteins in laser captured NFT's, 72 of which were identified by multiple unique peptides. Of these 72 proteins, 63 had previously unknown association with NFTs; one of these was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We validated by immunohistochemistry that GAPDH co-localized with the majority of NFTs as well as plaque-like structures in AD brain and was co-immunoprecipitated by antibodies to abnormal forms of tau in AD, but not tau from AD temporal cortex. Characterization of GAPDH showed that it, along with phosphorylated tau and Abeta peptides, was present in detergent-insoluble fractions from AD temporal cortex but not from age-matched controls. These data are the first proteomic investigation of NFTs. Moreover, our results validate this approach by demonstrating that GAPDH, a glycolytic and microtubule binding protein, not only co-localized to NFTs and immunoprecipitated with PHF-tau, but also is one of the few proteins known to undergo conversion to a detergent-insoluble form in AD.

Alzheimer Disease↗

PubMed2005

Microglial EP2 is critical to neurotoxicity from activated cerebral innate immunity.

Prostaglandin (PG) E(2) acts via four functionally antagonistic E-prostanoid (EP) receptors that are expressed on multiple cell types in the nervous system; these are designated EP1-4. We showed previously that EP2 null mice are protected from CD14-dependent neuronal damage in vivo following intracerebroventricular (ICV) injection of lipopolysaccharide (LPS). Clear interpretation of this neuroprotective outcome is limited because EP2 is expressed on glia and neurons. We tested the hypothesis that microglial EP2 is required for paracrine neurotoxicity following activation of innate immunity, using primary murine microglia and neuron co-cultures. We demonstrated that microglial EP2 was necessary for lipopolysaccharide (LPS)-activated microglia-mediated neurotoxicity, as well as induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Genetic deletion of microglial iNOS, pharmacological suppression of COX-2 activity, or addition of exogenous superoxide dismutase (SOD) and catalase in the presence of EP2 also abolished neurotoxicity. This loss of paracrine neurotoxicity by EP2(-/-) microglia occurred in the absence of reduced cytokine levels. We conclude that microglial EP2 is critical to innate immunity-mediated paracrine damage to neurons involving COX-2 and iNOS. EP2 should be considered as a therapeutic target for suppression of microglial innate immunity-mediated damage in neurodegenerative diseases.

Animals↗

PubMed2005

Role of glutathione in intracellular amyloid-alpha precursor protein/carboxy-terminal fragment aggregation and associated cytotoxicity.

Abstract Alterations in glutathione (GSH) metabolism are associated with neurodegeneration in Alzheimer's disease (AD), and GSH depletion follows application of exogenous fibrillar amyloid beta (Abeta) peptides in experimental systems; these results are commonly cited as evidence of oxidative damage in AD. We used MC65 human neuroblastoma cells that conditionally express carboxy-terminal fragments of the Abeta precursor protein (Abeta/CTFs) to directly test the hypothesis that GSH is part of the cellular response to stressors associated with Abeta/CTF accumulation and not simply a marker of oxidative damage. Our data showed that Abeta/CTFs accumulated by post-translational processes and were associated with progressive increases in oxidative damage and cytotoxicity. Ethycrinic acid (EA) or diethyl maleate (DEM), reagents that deplete GSH through non-specific thiol adduction, gave rise to dose-dependent cytotoxicity that was independent of Abeta/CTF expression and minimally responsive to alpha-tocopherol (AT). In contrast, buthionine sulfoximine (BSO), a selective inhibitor of GSH synthase, not only augmented Abeta/CTF-associated cell death but unexpectedly potentiated Abeta/CTF accumulation; both outcomes were completely suppressed by AT. These data suggest that antioxidants may serve as 'Abeta targeting' therapies that suppress toxic protein aggregation rather than simply acting as downstream radical scavengers.

Amyloid beta-Protein Precursor↗

PubMed2005

Microglial EP2 as a new target to increase amyloid beta phagocytosis and decrease amyloid beta-induced damage to neurons.

Epidemiologic and animal model data support a role for the prostaglandin pathway in AD pathogenesis. However, unexpected toxicity from protracted use of some nonsteroidal anti-inflammatory drugs (NSAIDs) compels investigation of therapeutic targets in this pathway other than COX inhibitors. Previously, we have shown that mice lacking one specific receptor for PGE2, EP2 (EP2-/-), are protected from the indirect neurotoxic effects of cerebral innate immune response mediated by CD14-dependent activation. Here we review data showing that EP2-/- microglia have a highly desirable combination of features: ablated indirect neurotoxicity following exposure to Abeta(1-42) coupled with enhanced phagocytosis of Abeta peptides, both synthetic and those deposited in human brain. These data point to microglial EP2 as a more focused target within the PG pathway for therapy in AD.

Alzheimer Disease↗

PubMed2005

Diminished taxol/GTP-stimulated tubulin polymerization in diseased region of brain from patients with late-onset or inherited Alzheimer's disease or frontotemporal dementia with parkinsonism linked to chromosome-17 but not individuals with mild cognitive impairment.

Neuronal microtubules are morphologically abnormal in diseased regions of brain from patients with late-onset Alzheimer's disease (LOAD). Here we tested the hypothesis that tubulin derived from gray matter of patients with multiple forms of dementia was functionally impaired. Following taxol/GTP stimulation of tubulin polymerization of gray matter extracts we observed reduced capacity of tubulin to polymerize in LOAD, but not individuals with mild cognitive impairment (MCI), compared to controls. Moreover, we observed similarly reduced taxol/GTP-stimulated tubulin polymerization from gray matter obtained from patients with AD caused by PSEN2 N141I mutation or frontotemporal dementia with parkinsonism linked to chromosome-17 caused (FTDP-17) by TAU V337M or P301L mutation. Our results show that modification of tubulin function may contribute to intermediate or late stages in the pathogenesis of sporadic and inherited AD as well as FTDP-17.

Aged↗

PubMed2005

Quantitative in vivo biomarkers of oxidative damage and their application to the diagnosis and management of Alzheimer's disease.

Dementia from Alzheimer's disease (AD) represents a significant and growing public health burden and presents a clear therapeutic imperative. Key to success in this undertaking will be biomarkers for the objective assessment of disease progression and response to therapeutics. Oxidative damage is one facet of AD pathogenesis for which there are experimentally validated quantitative in vivo biomarkers, the F2-isoprostanes (IsoPs). While plasma- or urine-based assays are desirable, consistent and reproducible cross-sectional data for increased F2-IsoPs in AD and mild cognitive impairment have been obtained only for CSF. In addition, measurement of CSF F2-IsoPs can increase the accuracy of laboratory-based classification of geriatric dementias, and have been used to assess objectively the response to anti-oxidant interventions in AD.

Alzheimer Disease↗

PubMed2004

Neuronal oxidative damage and dendritic degeneration following activation of CD14-dependent innate immune response in vivo.

The cause-and-effect relationship between innate immune activation and neurodegeneration has been difficult to prove in complex animal models and patients. Here we review findings from a model of direct innate immune activation via CD14 stimulation using intracerebroventricular injection of lipopolysaccharide. These data show that CD14-dependent innate immune activation in cerebrum leads to the closely linked outcomes of neuronal membrane oxidative damage and dendritic degeneration. Both forms of neuronal damage could be blocked by ibuprofen and alpha-tocopherol, but not naproxen or gamma-tocopherol, at pharmacologically relevant concentrations. This model provides a convenient method to determine effective agents and their appropriate dose ranges for protecting neurons from CD14-activated innate immunity-mediated damage, and can guide drug development for diseases, such as Alzheimer disease, that are thought to derive in part from CD14-activated innate immune response.

Journal Article↗

PubMed2004

Analysis of alpha-synuclein-associated proteins by quantitative proteomics.

To identify the proteins associated with soluble alpha-synuclein (AS) that might promote AS aggregation, a key event leading to neurodegeneration, we quantitatively compared protein profiles of AS-associated protein complexes in MES cells exposed to rotenone, a pesticide that produces parkinsonism in animals and induces Lewy body (LB)-like inclusions in the remaining dopaminergic neurons, and to vehicle. We identified more than 250 proteins associated with Nonidet P-40 soluble AS, and demonstrated that at least 51 of these proteins displayed significant differences in their relative abundance in AS complexes under conditions where rotenone was cytotoxic and induced formation of cytoplasmic inclusions immunoreactive to anti-AS. Overexpressing one of these proteins, heat shock protein (hsp) 70, not only protected cells from rotenone-mediated cytotoxicity but also decreased soluble AS aggregation. Furthermore, the protection afforded by hsp70 transfection appeared to be related to suppression of rotenone-induced oxidative stress as well as mitochondrial and proteasomal dysfunction.

Amino Acid Sequence↗

PubMed2004

Isoprostanes and related products of lipid peroxidation in neurodegenerative diseases.

Lipid peroxidation is a major outcome of free radical-mediated injury to brain, where it directly damages membranes and generates a number of oxidized products. Some of the chemically and metabolically stable oxidation products are useful in vivo biomarkers of lipid peroxidation. These include the isoprostanes (IsoPs) and isofurans (IsoFs), derived from arachidonic acid (AA), and neuroprostanes (NeuroPs), derived from docosahexaenoic acid (DHA). We have shown increased levels of IsoPs, NeuroPs, and IsoFs in diseased regions of brain from patients who died from advanced Alzheimer's disease (AD) or Parkinson's disease (PD). Increased cerebrospinal fluid (CSF) levels of IsoPs are present in patients with AD or Huntington's disease (HD) early in the course of their illness, and CSF IsoPs may improve the laboratory diagnostic accuracy for AD. In contrast, quantification of IsoPs in plasma and urine of AD patients has yielded inconsistent results. These results indicate that brain lipid peroxidation is a potential therapeutic target early in the course of AD and HD, that CSF IsoPs may aid in the assessment of anti-oxidant experimental therapeutics and laboratory diagnosis of AD.

Alzheimer Disease↗