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1.  Perivascular, but not Parenchymal, Cerebral Engraftment of Donor Cells after Non-Myeloablative Bone Marrow Transplantation 
Myeloablative (MyA) bone marrow transplantation (BMT) results in robust engraftment of BMT-derived cells in the central nervous system (CNS) and is neuroprotective in diverse experimental models of neurodegenerative diseases of brain and retina. However, MyA irradiation is associated with significant morbidity and mortality and does not represent a viable therapeutic option for the elderly. Non-myeloablative (NMyA) BMT is less toxic, but it is not known if the therapeutic efficacy observed with MyA BMT is preserved. As a first step to address this important gap in knowledge, we evaluated and compared engraftment characteristics of BMT-derived monocytes/microglia using several clinically relevant NMyA pretransplant conditioning regimens in C57BL/6 mice. These included chemotherapy (fludarabine and cyclophosphamide) with or without 2 Gy irradiation, and 5.5 Gy irradiation alone. Each regimen was followed by transplantation of whole bone marrow from green fluorescent protein-expressing wild type (wt) mice. While stable hematopoietic engraftment occurred, to varying degrees, in all NMyA regimens, only 5.5 Gy irradiation resulted in significant engraftment of BMT-derived cells in brain, where these cells were exclusively localized to perivascular, leptomeningeal, and related anatomic regions. Engraftment in retina under 5.5 Gy NMyA conditions was significantly reduced compared to MyA, but robust engraftment was identified in optic nerve. Advancing the therapeutic applications of BMT to neurodegenerative diseases will require identification of the barrier mechanisms MyA, but not NMyA, is able to overcome.
PMCID: PMC3706527  PMID: 23567123
2.  Ablation of the Microglial Protein DOCK2 Reduces Amyloid Burden in a Mouse Model of Alzheimer’s Disease 
Alzheimer’s disease (AD) neuropathology is characterized by innate immune activation primarily through prostaglandin E2 (PGE2) signaling. Dedicator of cytokinesis 2 (DOCK2) is a guanyl nucleotide exchange factor expressed exclusively in microglia in the brain and is regulated by PGE2 receptor EP2. DOCK2 modulates microglia cytokine secretion, phagocytosis, and paracrine neurotoxicity. EP2 ablation in experimental AD results in reduced oxidative damage and amyloid beta (Aβ) burden. This discovery led us to hypothesize that genetic ablation of DOCK2 would replicate the anti-Aβ effects of loss of EP2 in experimental AD. To test this hypothesis, we crossed mice that lacked DOCK2 (DOCK2−/−), were hemizygous for DOCK2 (DOCK2+/−), or that expressed two DOCK2 genes (DOCK2+/+) with APPswe-PS1Δe9 mice (a model of AD). While we found no DOCK2-dependent differences in cortex or in hippocampal microglia density or morphology in APPswe-PS1Δe9 mice, cerebral cortical and hippocampal Aβ plaque area and size were significantly reduced in 10-month-old APPswe-PS1Δe9/DOCK2−/− mice compared with APPswe-PS1Δe9/DOCK2+/+ controls. DOCK2 hemizygous APPswe-PS1Δe9 mice had intermediate Aβ plaque levels. Interestingly, soluble Aβ42 was not significantly different among the three genotypes, suggesting the effects were mediated specifically in fibrillar Aβ. In combination with earlier cell culture results, our in vivo results presented here suggest DOCK2 contributes to Aβ plaque burden via regulation of microglial innate immune function and may represent a novel therapeutic target for AD.
PMCID: PMC3602334  PMID: 23318649
innate immunity; microglia; amyloid beta; PGE2 receptor EP2; Alzheimer’s disease
3.  Antagonism of Neuronal Prostaglandin E2 Receptor Subtype 1 Mitigates Amyloid β Neurotoxicity In Vitro 
Multiple lines of evidence indicate that regional brain eicosanoid signaling is important in initiation and progression of neurodegenerative conditions that have a neuroinflammatory pathologic component, such as AD. We hypothesized that PGE2 receptor subtype 1 (EP1) signaling (linked to intracellular Ca2+ release) regulates Aβ peptide neurotoxicity and tested this in two complementary in vitro models: a human neuroblastoma cell line (MC65) producing Aβ1-40 through conditional expression of the APP C-terminal portion, and murine primary cortical neuron cultures exposed to Aβ1-42. In MC65 cells, EP1 receptor antagonist SC-51089 reduced Aβ neurotoxicity ~50% without altering high molecular weight Aβ immunoreactive species formation. Inositol-3-phosphate receptor antagonist 2-aminoethoxy-diphenyl borate offered similar protection. SC-51089 largely protected the neuron cultures from synthetic Aβ1-42 neurotoxicity. Nimodipine, a Ca2+ channel blocker, was completely neuroprotective in both models. Based on these data, we conclude that suppressing neuronal EP1 signaling may represent a promising therapeutic approach to ameliorate Aβ peptide neurotoxicity.
PMCID: PMC3700810  PMID: 22718277
4.  Anatomic and Clinical Pathology of Cognitive Impairment and Dementia 
Journal of Alzheimer's disease : JAD  2013;33(0 1):S181-S184.
Progressive cognitive impairment and its clinical culmination in dementia loom as a major public health problem in the coming generation of older adults, and this fact compels investigation to develop interventions that prevent, delay, or cure. The tools of anatomic pathology have provided key insights into the complex convergence of multiple diseases that commonly contribute to the dementia syndrome and its prodrome in the community setting, and they have suggested some exposures that may modulate disease burden. The tools of clinical pathology, in combination with neuroimaging, have revolutionized the approach to clinical investigation of Alzheimer's disease and are now doing the same with Lewy body disease and vascular brain injury. The tools of anatomic and clinical pathology will continue to contribute to our understanding of these diseases as we advance toward effective interventions for the diseases that commonly cause cognitive impairment and dementia in older adults.
PMCID: PMC3686298  PMID: 22699849
Alzheimer's disease; dementia; Lewy body disease; vascular brain injury
5.  Adult Changes in Thought Study: Dementia is an Individually Varying Convergent Syndrome with Prevalent Clinically Silent Diseases that may be Modified by Some Commonly Used Therapeutics 
Current Alzheimer research  2012;9(6):718-723.
The Adult Changes in Thought (ACT) study is a longitudinal population-based prospective cohort study of brain aging and incident dementia in the Seattle metropolitan area. Observational studies using autopsies from ACT indicate that dementia is a convergent syndrome that commonly derives from Alzheimer’s disease (AD), microvascular brain injury (μVBI), and Lewy body disease (LBD), and that these diseases have prevalent subclinical forms that also are commonly co-morbid. The existence of subclinical diseases highlights potential opportunities to intervene before the development of clinically apparent impairments. Our observations suggest that some such interventions already may exist to suppress processes of AD (statin therapy) or μVBI (treatment of hypertension). Reduced burden of LBD is associated with cigarette smoking; although smoking is not recommended as an intervention, these exposure data may provide clues to alternative neuroprotective mechanisms. Self reported anti-oxidant supplementation was without apparent effect in this cohort on indices of AD, μVBI, or LBD. Continued observational studies of brain aging will provide further insight into the convergent complexity of the dementia syndrome and its subclinical forms as well as highlight potential interventions that will require validation in clinical trials.
PMCID: PMC3409333  PMID: 22471861
6.  Apolipoprotein E isoforms and regulation of the innate immune response in brain of patients with Alzheimer’s disease 
Current Opinion in Neurobiology  2011;21(6):920-928.
The largest genetic risk for late-onset Alzheimer’s disease (AD) resides at the apolipoprotein E gene (APOE) locus, which has three common alleles (ε2, ε3, ε4) that encode three isoforms (apoE2, apoE3, apoE4). The very strong association of the APOE ε4 allele with AD risk and its role in the accumulation of amyloid β and animal models solidify the biological relevance of apoE isoforms but do not provide mechanistic insight. The innate immune response is consistently observed in AD and is a likely contributor to neuronal injury and response to injury. Here we review emerging data showing that apoE isoform regulation of multiple facets of the innate immune response in the brain may alter AD not only through amyloid β-dependent mechanisms, but also through other, amyloid β-independent mechanisms.
PMCID: PMC3237894  PMID: 21907569
7.  Role of Cerebrospinal Fluid and Plasma Biomarkers in the Diagnosis of Neurodegenerative Disorders and Mild Cognitive Impairment 
Biomarkers are one type of laboratory testing being developed in response to the therapeutic imperative for diseases that cause cognitive impairment and dementia. The role of biomarkers is already transforming the organization and conduct of clinical trials, and if successful will likely contribute in the future to the medical management of patients with these diseases. Despite the obvious utility of practicality of blood- or urine-based biomarkers, so far results from these fluid compartments have not been reproducible. In contrast, substantial progress has been made in cerebrospinal fluid biomarkers. Here we review the stages of cerebrospinal fluid biomarker development for several common and unusual diseases that cause cognitive impairment and dementia, stressing the distinction between diagnostic and mechanistic biomarkers. Future applications will likely focus on diagnosis of latent or early-stage disease, assessment of disease progression, mechanism of injury, and response to experimental therapeutics.
PMCID: PMC3213691  PMID: 21725901
Alzheimer’s disease; Parkinson’s disease; vascular brain injury; biomarkers; cerebrospinal fluid; neurodegenerative disorders; mild cognitive impairment
8.  Apolipoprotein C-I is an APOE genotype-dependent suppressor of glial activation 
Inheritance of the human ϵ4 allele of the apolipoprotein (apo) E gene (APOE) significantly increases the risk of developing Alzheimer’s disease (AD), in addition to adversely influencing clinical outcomes of other neurologic diseases. While apoE isoforms differentially interact with amyloid β (Aβ), a pleiotropic neurotoxin key to AD etiology, more recent work has focused on immune regulation in AD pathogenesis and on the mechanisms of innate immunomodulatory effects associated with inheritance of different APOE alleles. APOE genotype modulates expression of proximal genes including APOC1, which encodes a small apolipoprotein that is associated with Aβ plaques. Here we tested the hypothesis that APOE-genotype dependent innate immunomodulation may be mediated in part by apoC-I.
ApoC-I concentration in cerebrospinal fluid from control subjects of differing APOE genotypes was quantified by ELISA. Real-time PCR and ELISA were used to analyze apoC-I mRNA and protein expression, respectively, in liver, serum, cerebral cortex, and cultured primary astrocytes derived from mice with targeted replacement of murine APOE for human APOE ϵ3 or ϵ4. ApoC-I direct modulation of innate immune activity was investigated in cultured murine primary microglia and astrocytes, as well as human differentiated macrophages, using specific toll-like receptor agonists LPS and PIC as well as Aβ.
ApoC-I levels varied with APOE genotype in humans and in APOE targeted replacement mice, with ϵ4 carriers showing significantly less apoC-I in both species. ApoC-I potently reduced pro-inflammatory cytokine secretion from primary murine microglia and astrocytes, and human macrophages, stimulated with LPS, PIC, or Aβ.
ApoC-I is immunosuppressive. Our results illuminate a novel potential mechanism for APOE genotype risk for AD; one in which patients with an ϵ4 allele have decreased expression of apoC-I resulting in increased innate immune activity.
PMCID: PMC3490924  PMID: 22883744
ApoE; ApoC-I; Alzheimer’s disease; Cerebrospinal fluid; Targeted replacement mice
9.  Ecology of aging human brain 
Archives of neurology  2011;68(8):1049-1056.
Alzheimer’s disease (AD), cerebral vascular brain injury (VBI), and isocortical Lewy body (LB) disease (LBD) are the major contributors to dementia in community- or population-based studies: Adult Changes in Thought (ACT) study, Honolulu-Asia Aging Study (HAAS), Nun Study (NS), and Oregon Brain Aging Study (OBAS). However, the prevalence of clinically silent forms of these diseases in cognitively normal (CN) adults is less clear.
We evaluated 1672 brain autopsies from ACT, HAAS, NS, and OBAS of which 424 met criteria for CN.
Of these, 336 cases had a comprehensive neuropathologic examination of neuritic plaque (NP) density, Braak stage for neurofibrillary tangles (NFTs), Lewy body (LB) distribution, and number of cerebral microinfarcts (CMIs).
47% of CN cases had moderate or frequent NP density; of these 6% also had Braak stage V or VI for NFTs. 15% of CN cases had medullary LBD; 8% also had nigral and 4% isocortical LBD. The presence of any CMIs was identified in 33% and high level CMIs in 10% of CN individuals. Overall burden of lesions in each individual and their co-morbidity varied widely within each study but were similar among studies.
These data show an individually varying complex convergence of subclinical diseases in the brain of older CN adults. Appreciating this ecology should help guide future biomarker or neuroimaging studies as well as clinical trials that focus on community- or population-based cohorts.
PMCID: PMC3218566  PMID: 21825242
Alzheimer’s disease; vascular brain injury; Lewy body disease; cognitive aging
10.  Increased Cerebrospinal Fluid F2-Isoprostanes are Associated with Aging and Latent Alzheimer’s Disease as Identified by Biomarkers 
Neuromolecular medicine  2010;13(1):37-43.
Alzheimer’s disease (AD) is a common age-related chronic illness with latent, prodrome, and fully symptomatic dementia stages. Increased free radical injury to regions of brain is one feature of prodrome and dementia stages of AD; however, it also is associated with advancing age. This raises the possibility that age-related free radical injury to brain might be caused in part or in full by latent AD. We quantified free radical injury in the central nervous system with cerebrospinal fluid (CSF) F2-isoprostanes (IsoPs) in 421 clinically normal individuals and observed a significant increase over the adult human lifespan (P < 0.001). Using CSF amyloid (A) β42 and tau, we defined normality using results from 28 clinically normal individuals < 50 years old, and then stratified 74 clinically normal subjects ≥ 60 years into those with CSF that had normal CSF Aβ42 and tau (n=37); abnormal CSF Aβ42 and tau, the biomarker signature of AD (n=24); decreased Aβ42 only (n=4); or increased tau only (n=9). Increased CSF F2-IsoPs were present in clinically normal subjects with the biomarker signature of AD (P < 0.05) and those subjects with increased CSF tau (P < 0.001). Finally, we analyzed the relationship between age and CSF F2-IsoPs for those clinically normal adults with normal CSF (n=37) and those with abnormal CSF Aβ42 and/or tau (n=37); only those with normal CSF demonstrated a significant increase with age (P < 0.01). These results show that CSF F2-IsoPs increased across the human lifespan and that this age-related increase in free radical injury to brain persisted after culling those with laboratory evidence of latent AD.
PMCID: PMC3000441  PMID: 20632131
Alzheimer’s disease; cerebrospinal fluid; biomarkers; Aβ42; tau; F2-isoprostanes
11.  Mitochondrial therapeutics in Alzheimer's disease and Parkinson's disease 
In neurons, mitochondria serve a wide variety of processes that are integral to their function and survival. It is, therefore, not surprising that evidence of mitochondrial dysfunction is observed across numerous neurodegenerative diseases. Alzheimer's disease and Parkinson's disease are two such diseases in which aberrant mitochondrial activity is proposed to contribute to pathogenesis. Current therapies for each disease target various mechanisms, but few, if any, directly target improved mitochondrial function. Recent discoveries pertaining to mitochondrial dynamics reveal that regulation of mitochondrial fission and fusion may play a key role in the pathogenesis of these diseases and consequently could be novel future therapeutic targets.
PMCID: PMC3226310  PMID: 21722346
12.  Pathologic correlates of dementia in individuals with Lewy body disease 
Cognitive impairment and dementia are more common in patients with Parkinson disease (PD) than age-matched controls, and appear to become more frequent as PD progresses. However, estimates of dementia in patients with PD have varied widely, likely due in part to differences in case definition, case ascertainment, and methodology. First, we review investigations of usual pathologic correlates of dementia in patients with brainstem (b) Lewy Body Disease (LBD) and report our findings from the initial 266 brain autopsies from a population-based study of brain aging and incident dementia. Our results showed that 2.6% of subjects were diagnosed with PD during life but that 20% had bLBD at autopsy. Seventy percent of individuals with bLBD had high-level of one or more cerebral pathologic changes significantly associated with dementia: Alzheimer's disease (AD), cerebral (c) LBD, or microvascular brain injury (μVBI); these were commonly co-morbid. Next we consider proposed contributors to cognitive impairment and dementia in the approximately 30% of patients with only bLBD, including regionally selective dendritic degeneration of neostriatal medium spiny neurons. Diseases contributing to cognitive impairment and dementia in patients with bLBD are heterogeneous, providing diagnostic challenges as well as multiple opportunities for successful intervention in patients with PD.
PMCID: PMC2881486  PMID: 20522091
13.  Cerebrospinal Fluid Biomarkers in Mild Cognitive Impairment and Dementia 
Given the magnitude of the public health problem of dementia in the elderly, there is a pressing need for research, development, and timely application of biomarkers that will identify latent and prodromal illness as well as dementia. Although identification of risk factors and neuroimaging measures will remain key to these efforts, this review focuses on recent progress in the discovery, validation, and standardization of cerebrospinal fluid (CSF) biomarkers, small molecules and macromolecules whose CSF concentration can aid in diagnosis at different stages of disease as well as in assessment of disease progression and response to therapeutics. A multimodal approach that brings independent information from risk factor assessment, neuroimaging, and biomarkers may soon guide physicians in the early diagnosis and management of cognitive impairment in the elderly.
PMCID: PMC2891184  PMID: 20061646
Alzheimer’s disease; biomarkers; cerebrospinal fluid; Lewy body disease; vascular cognitive impairment
14.  Association between lifetime cigarette smoking and Lewy body accumulation 
Cigarette smoking has been associated repeatedly in observational studies with decreased risk of Parkinson's disease (PD), but its relationship to the risk of dementia or Alzheimer's disease (AD) is inconsistent. All of these studies have used clinical diagnoses of disease. We tested the hypothesis that lifetime cigarette use might be associated with reduced risk of neuropathologic changes of Lewy-related pathology (LRP) in multiple brain regions or with reduced risk of consensus neuropathologic changes of AD in a prospective community-based study of brain aging and dementia, the Adult Changes in Thought (ACT) study. We observed that heavy lifetime cigarette smoking (> 50 pack years) was associated with significantly reduced relative risk (RR) for LRP, but not AD-type pathologic changes, after correcting for selection bias, and with significantly reduced frequency of LRP in the substantia nigra. These findings are the first of which we are aware to associate reduced LRP in human brain with any exposure, and substantiate observational studies that have associated cigarette smoking with reduced risk of PD. Although cigarette smoking is too toxic to suggest as a treatment, if confirmed, these findings may guide future therapeutic strategies that attempt to suppress LRP in human brain by other means.
PMCID: PMC2864364  PMID: 19531100
Alzheimer's disease; Lewy body; smoking; neuropathology
15.  Cognitive Impairment and Dementia in Patients with Parkinson Disease 
Parkinson disease (PD) is an already prevalent neurodegenerative disease that is poised to at least double over the next 25 years. Although best known for its characteristic movement disorder, PD is now appreciated commonly to cause cognitive impairment, including dementia, and behavioral changes. Dementia in patients with PD is consequential and has been associated with reduced quality of life, shortened survival, and increased caregiver distress. Here we review clinical presentation and progression, pathological bases, identification of genetic risk factors, development of small molecule biomarkers, and emerging treatments for cognitive impairment in patients with PD.
PMCID: PMC2804995  PMID: 19754405
16.  Biomarkers for cognitive impairment and dementia in elderly people 
Lancet neurology  2008;7(8):704-714.
The threat of a looming pandemic of dementia in elderly people highlights the compelling need for the development and validation of biomarkers that can be used to identify pre-clinical and prodromal stages of disease in addition to fully symptomatic dementia. Although predictive risk factors and correlative neuroimaging measures will have important roles in these efforts, this Review describes recent progress in the discovery, validation, and standardization of molecular biomarkers – small molecules and macromolecules whose concentration in brain or biological fluids can aid in diagnosis at different stages of the more common dementing diseases and in the assessment of disease progression and response to therapeutics. An approach that efficiently combines independent information from risk factor assessment, neuroimaging measures, and biomarkers may soon guide clinicians in the early diagnosis and management of cognitive impairment in elderly people.
PMCID: PMC2710375  PMID: 18635019
17.  Proteomics of Human Neurodegenerative Diseases 
The technology, experimental approaches, and bioinformatics that support proteomic research are evolving rapidly. The application of these new capabilities to the study of neurodegenerative diseases is providing insight into the biochemical pathogenesis of neurodegeneration as well as fueling major efforts in biomarker discovery. Here, we review the fundamentals of commonly used proteomic approaches and the outcomes of these investigations with autopsy and cerebrospinal fluid samples from patients with neurodegenerative diseases.
PMCID: PMC2710115  PMID: 18800015
Alzheimer disease; Cerebrospinal fluid; Mass spectrometry; Neurodegeneration; Parkinson disease; Proteomics
18.  Therapeutic targets in prostaglandin E2 signaling for neurologic disease 
Current medicinal chemistry  2008;15(19):1863-1869.
Prostaglandins (PGs) are potent autocrine and paracrine oxygenated lipid molecules that contribute appreciably to physiologic and pathophysiologic responses in almost all organs, including brain. Emerging data indicate that the PGs, and more specifically PGE2, play a central role in brain diseases including ischemic injury and several neurodegenerative diseases. Given concerns over the potential toxicity from protracted use of cyclooxygenase inhibitors in the elderly, attention is now focused on blocking PGE2 signaling that is mediated by interactions with four distinct G protein-coupled receptors, EP1-4, which are differentially expressed on neuronal and glial cells throughout the central nervous system. EP1 activation has been shown to mediate Ca2+-dependent neurotoxicity in ischemic injury. EP2 activation has been shown to mediate microglial-induced paracrine neurotoxicity as well as suppress microglia internalization of aggregated neurotoxic peptides. Animal models support the potential efficacy of targeting specific EP receptor subtypes in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and ischemic stroke. However promising these preclinical studies are, they have yet to be followed by clinical trials targeting any EP receptor in neurologic diseases.
PMCID: PMC2693919  PMID: 18691044
Prostaglandins; PGE2; CNS; neurodegeneration; EP receptors
19.  Identification of novel proteins affected by rotenone in mitochondria of dopaminergic cells 
BMC Neuroscience  2007;8:67.
Many studies have shown that mitochondrial dysfunction, complex I inhibition in particular, is involved in the pathogenesis of Parkinson's disease (PD). Rotenone, a specific inhibitor of mitochondrial complex I, has been shown to produce neurodegeneration in rats as well as in many cellular models that closely resemble PD. However, the mechanisms through which complex I dysfunction might produce neurotoxicity are as yet unknown. A comprehensive analysis of the mitochondrial protein expression profile affected by rotenone can provide important insight into the role of mitochondrial dysfunction in PD.
Here, we present our findings using a recently developed proteomic technology called SILAC (stable isotope labeling by amino acids in cell culture) combined with polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry to compare the mitochondrial protein profiles of MES cells (a dopaminergic cell line) exposed to rotenone versus control. We identified 1722 proteins, 950 of which are already designated as mitochondrial proteins based on database search. Among these 950 mitochondrial proteins, 110 displayed significant changes in relative abundance after rotenone treatment. Five of these selected proteins were further validated for their cellular location and/or treatment effect of rotenone. Among them, two were confirmed by confocal microscopy for mitochondrial localization and three were confirmed by Western blotting (WB) for their regulation by rotenone.
Our findings represent the first report of these mitochondrial proteins affected by rotenone; further characterization of these proteins may shed more light on PD pathogenesis.
PMCID: PMC2000881  PMID: 17705834
20.  Prostaglandin E2 receptor subtype 2 (EP2) regulates microglial activation and associated neurotoxicity induced by aggregated α-synuclein 
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 α-synuclein potently activates microglia and causes neurotoxicity. However, the mechanisms by which aggregated α-synuclein activates microglia are not understood fully.
We investigated the role of prostaglandin E2 receptor subtype 2 (EP2) in α-synuclein aggregation-induced microglial activation using ex vivo, in vivo and in vitro experimental systems.
Results demonstrated that ablation of EP2(EP2-/-) significantly enhanced microglia-mediated ex vivo clearance of α-synuclein aggregates (from mesocortex of Lewy body disease patients) while significantly attenuating neurotoxicity and extent of α-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 α-synuclein.
Thus, it appears that microglial EP2 plays a critical role in α-synuclein-mediated neurotoxicity.
PMCID: PMC1766347  PMID: 17204153
21.  Apolipoprotein E isoform-dependent dendritic recovery of hippocampal neurons following activation of innate immunity 
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.
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.
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.
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.
PMCID: PMC1584222  PMID: 16934151
22.  Apolipoprotein E-specific innate immune response in astrocytes from targeted replacement mice 
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 β 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 ε4 allele (TR APOE4) and that derives from p38 mitogen-activated protein kinase (p38MAPK) activity.
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-κB) subunit activity were measured and compared.
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-κB subunit activity.
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-κB signaling in these two cell types.
PMCID: PMC1502133  PMID: 16603079
23.  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.
PMCID: PMC527876  PMID: 15498098

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