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1.  Immune Heterogeneity in Neuroinflammation: Dendritic Cells in the Brain 
Dendritic cells (DC) are critical to an integrated immune response and serve as the key link between the innate and adaptive arms of the immune system. Under steady state conditions, brain DC’s act as sentinels, continually sampling their local environment. They share this function with macrophages derived from the same basic hemopoietic (bone marrow-derived) precursor and with parenchymal microglia that arise from a unique non-hemopoietic origin. While multiple cells may serve as antigen presenting cells (APCs), dendritic cells present both foreign and self-proteins to naïve T cells that, in turn, carry out effector functions that serve to protect or destroy. The resulting activation of the adaptive response is a critical step to resolution of injury or infection and is key to survival. In this review we will explore the critical roles that DCs play in the brain’s response to neuroinflammatory disease with emphasis on how the brain’s microenvironment impacts these actions.
PMCID: PMC4279719  PMID: 23114889
Neuroinflammation; Dendritic cells; Immunosuppression; Microglia; T-cells
2.  The impact of human and mouse differences in NOS2 gene expression on the brain’s redox and immune environment 
Mouse models are used in the study of human disease. Despite well-known homologies, the difference in immune response between mice and humans impacts the application of data derived from mice to human disease outcomes. Nitric oxide synthase-2 (NOS2) is a key gene that displays species-specific outcomes via altered regulation of the gene promoter and via post-transcriptional mechanisms in humans that are not found in mice. The resulting levels of NO produced by activation of human NOS2 are different from the levels of NO produced by mouse Nos2. Since both tissue redox environment and immune responsiveness are regulated by the level of NO and its interactions, we investigated the significance of mouse and human differences on brain oxidative stress and on immune activation in HuNOS2tg/mNos2-/- mice that express the entire human NOS2 gene and that lack a functional mNos2 compared to wild type (WT) mice that express normal mNos2.
Similarly to human, brain tissue from HuNOS2tg/mNos2-/- mice showed the presence of a NOS2 gene 3′UTR binding site. We also identified miRNA-939, the binding partner for this site, in mouse brain lysates and further demonstrated reduced levels of nitric oxide (NO) typical of the human immune response on injection with lipopolysaccharide (LPS). HuNOS2tg/mNos2-/- brain samples were probed for characteristic differences in redox and immune gene profiles compared to WT mice using gene arrays. Selected genes were also compared against mNos2-/- brain lysates. Reconstitution of the human NOS2 gene significantly altered genes that encode multiple anti-oxidant proteins, oxidases, DNA repair, mitochondrial proteins and redox regulated immune proteins. Expression levels of typical pro-inflammatory, anti-inflammatory and chemokine genes were not significantly different with the exception of increased TNFα and Ccr1 mRNA expression in the HuNOS2tg/mNos2-/- mice compared to WT or mNos2-/- mice.
NO is a principle factor in establishing the tissue redox environment and changes in NO levels impact oxidative stress and immunity, both of which are primary characteristics of neurodegenerative diseases. The HuNOS2tg/mNos2-/- mice provide a potentially useful mechanism to address critical species- specific immune differences that can impact the study of human diseases.
PMCID: PMC4247207  PMID: 25403885
NOS2; Mouse models; Neurodegeneration; Redox; Inflammation; Nitric oxide
3.  Longitudinal study of differential protein expression in an Alzheimer’s mouse model lacking inducible nitric oxide synthase 
Journal of proteome research  2013;12(10):10.1021/pr4005103.
Alzheimer’s disease (AD) is a complex neurodegenerative process that involves altered brain immune, neuronal and metabolic functions. Understanding the underlying mechanisms has relied on mouse models that mimic components of AD pathology. We used gel-free, label-free LC/MS/MS to quantify protein and phosphopeptide levels in brains of APPSwDI/NOS2−/− (CVN-AD) mice. CVN-AD mice show a full spectrum of AD-like pathology, including amyloid deposition, hyperphosphorylated and aggregated tau and neuronal loss that worsens with age. Tryptic digests, with or without phosphopeptide enrichment on an automated titanium dioxide LC system, were separated by on-line two-dimensional LC and analyzed on a Waters Synapt G2 HDMS, yielding relative expression for >950 proteins and >1100 phosphopeptides. Among differentially expressed proteins were known markers found in humans with AD, including GFAP and C1Q. Phosphorylation of connexin 43, not previously described in AD, was increased at 42 weeks, consistent with dysregulation of gap junctions and activation of astrocytes. Additional alterations in phosphoproteins suggests dysregulation of mitochondria, synaptic transmission, vesicle trafficking and innate immune pathways. These data validate the CVN-AD mouse model of AD, identify novel disease and age-related changes in the brain during disease progression and demonstrate the utility of integrating unbiased and phosphoproteomics for understanding disease processes in AD.
PMCID: PMC3875406  PMID: 24006891
Alzheimer’s disease; phosphorylation; NOS2; proteomics; mass spectrometry; neurodegeneration
4.  Elevated copper in the amyloid plaques and iron in the cortex are observed in mouse models of Alzheimer’s disease that exhibit neurodegeneration 
In Alzheimer’s disease (AD), alterations in metal homeostasis, including the accumulation of metal ions in the plaques and an increase of iron in the cortex, have been well documented but the mechanisms involved are poorly understood.
In this study, we compared the metal content in the plaques and the iron speciation in the cortex of three mouse models, two of which show neurodegeneration (5xFAD and Tg-SwDI/NOS2−/− (CVN) and one that shows very little neurodegeneration (PSAPP).
The Fe, Cu, and Zn contents and speciation were determined using synchrotron X-ray fluorescence microscopy (XFM) and X-ray absorption spectroscopy (XAS), respectively.
In the mouse models with reported significant neurodegeneration, we found that plaques contained ~25% more copper compared to the PSAPP mice. The iron content in the cortex increased at the late stage of the disease in all mouse models, but iron speciation remains unchanged.
The elevation of copper in the plaques and iron in the cortex is associated with AD severity, suggesting that these redox-active metal ions may be inducing oxidative damage and directly influencing neurodegeneration.
PMCID: PMC4051362  PMID: 24926425
Alzheimer’s disease; amyloid plaques; copper; iron; transgenic mice; X-ray fluorescence microscopy; X-ray absorption spectroscopy; infrared microscopy
5.  Microglial-neuronal interactions during neurodegenerative diseases 
The impact of the brain’s immune response to disease is no longer relegated to a secondary event of limited interest. Multiple types of brain and spinal cord injuries and diseases show varying immune phenotypes over the time course of the disease. The collection of articles illustrates the wide reach of brain immunity and provides research papers and reviews that illustrate this point. These articles have added both new information and new directions to consider, and I would like to thank all of the participants for their insightful contributions to the field.
PMCID: PMC3652666  PMID: 23404094
6.  Accelerating drug discovery for Alzheimer's disease: best practices for preclinical animal studies 
Animal models have contributed significantly to our understanding of the underlying biological mechanisms of Alzheimer's disease (AD). As a result, over 300 interventions have been investigated and reported to mitigate pathological phenotypes or improve behavior in AD animal models or both. To date, however, very few of these findings have resulted in target validation in humans or successful translation to disease-modifying therapies. Challenges in translating preclinical studies to clinical trials include the inability of animal models to recapitulate the human disease, variations in breeding and colony maintenance, lack of standards in design, conduct and analysis of animal trials, and publication bias due to under-reporting of negative results in the scientific literature. The quality of animal model research on novel therapeutics can be improved by bringing the rigor of human clinical trials to animal studies. Research communities in several disease areas have developed recommendations for the conduct and reporting of preclinical studies in order to increase their validity, reproducibility, and predictive value. To address these issues in the AD community, the Alzheimer's Drug Discovery Foundation partnered with Charles River Discovery Services (Morrisville, NC, USA) and Cerebricon Ltd. (Kuopio, Finland) to convene an expert advisory panel of academic, industry, and government scientists to make recommendations on best practices for animal studies testing investigational AD therapies. The panel produced recommendations regarding the measurement, analysis, and reporting of relevant AD targets, th choice of animal model, quality control measures for breeding and colony maintenance, and preclinical animal study design. Major considerations to incorporate into preclinical study design include a priori hypotheses, pharmacokinetics-pharmacodynamics studies prior to proof-of-concept testing, biomarker measurements, sample size determination, and power analysis. The panel also recommended distinguishing between pilot 'exploratory' animal studies and more extensive 'therapeutic' studies to guide interpretation. Finally, the panel proposed infrastructure and resource development, such as the establishment of a public data repository in which both positive animal studies and negative ones could be reported. By promoting best practices, these recommendations can improve the methodological quality and predictive value of AD animal studies and make the translation to human clinical trials more efficient and reliable.
PMCID: PMC3218805  PMID: 21943025
7.  Lithium Treatment of APPSwDI/NOS2−/− Mice Leads to Reduced Hyperphosphorylated Tau, Increased Amyloid Deposition and Altered Inflammatory Phenotype 
PLoS ONE  2012;7(2):e31993.
Lithium is an anti-psychotic that has been shown to prevent the hyperphosphorylation of tau protein through the inhibition of glycogen-synthase kinase 3-beta (GSK3β). We recently developed a mouse model that progresses from amyloid pathology to tau pathology and neurodegeneration due to the genetic deletion of NOS2 in an APP transgenic mouse; the APPSwDI/NOS2−/− mouse. Because this mouse develops tau pathology, amyloid pathology and neuronal loss we were interested in the effect anti-tau therapy would have on amyloid pathology, learning and memory. We administered lithium in the diets of APPSwDI/NOS2−/− mice for a period of eight months, followed by water maze testing at 12 months of age, immediately prior to sacrifice. We found that lithium significantly lowered hyperphosphorylated tau levels as measured by Western blot and immunocytochemistry. However, we found no apparent neuroprotection, no effect on spatial memory deficits and an increase in histological amyloid deposition. Aβ levels measured biochemically were unaltered. We also found that lithium significantly altered the neuroinflammatory phenotype of the brain, resulting in enhanced alternative inflammatory response while concurrently lowering the classical inflammatory response. Our data suggest that lithium may be beneficial for the treatment of tauopathies but may not be beneficial for the treatment of Alzheimer's disease.
PMCID: PMC3276493  PMID: 22347510
8.  Diverse inflammatory responses in transgenic mouse models of Alzheimer's disease and the effect of immunotherapy on these responses 
ASN NEURO  2011;3(5):e00069.
While the presence of an inflammatory response in AD (Alzheimer's disease) is well known, the data on inflammation are conflicting, suggesting that inflammation either attenuates pathology, exacerbates it or has no effect. Our goal was to more fully characterize the inflammatory response in APP (amyloid precursor protein) transgenic mice with and without disease progression. In addition, we have examined how anti-Aβ (amyloid β-peptide) immunotherapy alters this inflammatory response. We have used quantitative RT–PCR (reverse transcription–PCR) and protein analysis to measure inflammatory responses ranging from pro-inflammatory to anti-inflammatory and repair factors in transgenic mice that develop amyloid deposits only (APPSw) and amyloid deposits with progression to tau pathology and neuron loss [APPSw/NOS2−/− (nitric oxide synthase 2−/−)]. We also examined tissues from previously published immunotherapy studies. These studies were a passive immunization study in APPSw mice and an active vaccination study in APPSw/NOS2−/− mice. Both studies have already been shown to lower amyloid load and improve cognition. We have found that amyloid deposition is associated with high expression of alternative activation and acquired deactivation genes and low expression of pro-inflammatory genes, whereas disease progression is associated with a mixed phenotype including increased levels of some classical activation factors. Immunotherapy targeting amyloid deposition in both mouse models resulted in decreased alternative inflammatory markers and, in the case of passive immunization, a transient increase in pro-inflammatory markers. Our results suggest that an alternative immune response favours retention of amyloid deposits in the brain, and switching away from this state by immunotherapy permits removal of amyloid.
PMCID: PMC3227004  PMID: 21995345
alternative activation; amyloid deposition; immunotherapy; microglia; neuroinflammation; Aβ, amyloid β-peptide; AD, Alzheimer's disease; AG1, arginase 1; APP, amyloid precursor protein; IL-1β, interleukin 1β; LPS, lipopolysaccharide; MMP, matrix metalloprotease; MR, mannose receptor; Mrc1, mannose receptor C1; NFT, neurofibrillary tangle; NOS2, nitric oxide synthase 2; RT–PCR, reverse transcription–PCR; SPHK, sphingosine kinase; TGFβ, transforming growth factor β; TNFα, tumour necrosis factor α; WT, wild-type
9.  Enhanced Capillary Amyloid Angiopathy Associated Pathology in Tg-SwDI Mice with Deleted Nitric Oxide Synthase 2 
Stroke; a journal of cerebral circulation  2010;41(10 Suppl):S135-S138.
Cerebral amyloid angiopathy (CAA)-type 1 is characterized by amyloid β-protein (Aβ) deposition along cerebral capillaries and is accompanied by perivascular neuroinflammation and accumulation of phospho-tau protein. Tg-SwDI mice recapitulate capillary amyloid deposition and associated neuroinflammation but lack accmulation of perivascular phospho-tau protein.
Tg-SwDI mice were bred onto a nitric oxide synthase 2 gene knockout (NOS2−/−) background and aged for one year. Brains were harvested and analyzed using immunohistochemical and quantitative stereological methods to determine the extent of capillary amyloid deposition, perivascular activated microglia, and cell-specific accumulation of phospho-tau protein. Similar methods were also used to compare Tg-SwDI/NOS2−/− and human CAA-type 1 brain tissues.
The absence of NOS2 had no effect on the regional pattern or frequency of capillary CAA or the numbers of perivascular activated microglia in Tg-SwDI mice. On the other hand, Tg-SwDI/NOS2−/− mice accumulated phospho-tau protein in perivascular neurons and activated microglia. Tg-SwDI/NOS2−/− mice exhibited a very similar distribution of capillary amyloid, activated microglia, and perivascular phospho-tau protein as seen in human CAA-type 1.
These findings indicate that Tg-SwDI/NOS2−/− mice more fully recapitulate the pathological changes observed with capillary amyloid in human CAA-type 1.
PMCID: PMC2997647  PMID: 20876489
capillary; cerebral amyloid angiopathy; nitric oxide synthase 2; phospho-tau; transgenic mice
10.  Activation of matrix metalloproteinases following anti-Aβ immunotherapy; implications for microhemorrhage occurrence 
Anti-Aβ immunotherapy is a promising approach to the prevention and treatment of Alzheimer's disease (AD) currently in clinical trials. There is extensive evidence, both in mice and humans that a significant adverse event is the occurrence of microhemorrhages. Also, vasogenic edema was reported in phase 2 of a passive immunization clinical trial. In order to overcome these vascular adverse effects it is critical that we understand the mechanism(s) by which they occur.
We have examined the matrix metalloproteinase (MMP) protein degradation system in two previously published anti-Aβ immunotherapy studies. The first was a passive immunization study in which we examined 22 month old APPSw mice that had received anti-Aβ antibodies for 1, 2 or 3 months. The second is an active vaccination study in which we examined 16 month old APPSw/NOS2-/- mice treated with Aβ vaccination for 4 months.
There is a significant activation of the MMP2 and MMP9 proteinase degradation systems by anti-Aβ immunotherapy, regardless of whether this is delivered through active vaccination or passive immunization. We have characterized this activation by gene expression, protein expression and zymography assessment of MMP activity.
Since the MMP2 and MMP9 systems are heavily implicated in the pathophysiology of intracerbral hemorrhage, these data may provide a potential mechanism of microhemorrhage due to immunotherapy. Increased activity of the MMP system, therefore, is likely to be a major factor in increased microhemorrhage occurrence.
PMCID: PMC3182918  PMID: 21906275
Immunotherapy; matrix metalloproteinases; inflammation; microhemorrhage; amyloid; cerebral amyloid angiopathy; transgenic mouse; Alzheimer's disease
11.  Interaction of NG2+ glial progenitors and microglia/macrophages from the injured spinal cord 
Glia  2010;58(4):410-422.
Spinal cord contusion produces a central lesion surrounded by a peripheral rim of residual white matter. Despite stimulation of NG2+ progenitor cell proliferation, the lesion remains devoid of normal glia chronically after spinal cord injury (SCI). To investigate potential cell-cell interactions of the predominant cells in the lesion at 3 days after injury, we used magnetic activated cell sorting to purify NG2+ progenitors and OX42+ microglia/macrophages from contused rat spinal cord. Purified NG2+ cells from the injured cord grew into spherical masses when cultured in defined medium with FGF2 plus GGF2. The purified OX42+ cells did not form spheroids and significantly reduced sphere growth by NG2+ cells in co-cultures. Conditioned medium from these OX42+ cells, unlike that from normal peritoneal macrophages or astrocytes also inhibited growth of NG2+ cells, suggesting inhibition by secreted factors. Expression analysis of freshly purified OX42+ cells for a panel of 6 genes for secreted factors showed expression of several that could contribute to inhibition of NG2+ cells. Further, the pattern of expression of four of these, TNFα, TSP1, TIMP1, MMP9, in sequential coronal tissue segments from a 2 cm length of cord centered on the injury epicenter correlated with the expression of Iba1, a marker gene for OX42+ cells, strongly suggesting a potential regional influence by activated microglia/macrophages on NG2+ cells in vivo after SCI. Thus, the non-replacement of lost glial cells in the central lesion zone may involve, at least in part, inhibitory factors produced by microglia/macrophages that are concentrated within the lesion.
PMCID: PMC2807472  PMID: 19780197
glial progenitors; NG2; macrophage; proliferation; spinal cord injury; cell culture; MACS
12.  APOE genotype-specific differences in the innate immune response 
Neurobiology of aging  2007;30(9):1350-1360.
Apolipoprotein-E protein is an endogenous immunomodulatory agent that affects both the innate and the adaptive immune responses. Since individuals with the APOE4 gene demonstrate worsened pathology and poorer outcomes in many neurological disorders, we examined isoform specific differences in the response of microglia, the primary cellular component of the brain’s innate immune response, in detail. Our data demonstrate that microglia derived from APOE4/4 targeted replacement mice demonstrate a pro-inflammatory phenotype that includes altered cell morphology, increased NO production associated with increased NOS2 mRNA levels, and higher pro-inflammatory cytokine production (TNFα, IL-6, IL12p40) compared to microglia derived from APOE3/3 targeted replacement mice. The effect is gene dose dependent and increases with the number of APOE4 gene alleles. The APOE genotype-specific immune profile observed in the microglial immune response is also observed in the cortex of aged APOE3/3 and APOE4/4 mice treated with lipopolysacchride (LPS) and in peripheral (peritoneal) macrophages. To determine if APOE4’s action resulted from an isoform specific difference in effective levels of the apolipoproteins, we generated mice expressing only a single allele of APOE3. Immune-stimulated macrophages from APOE3/0 mice demonstrated an increased inflammatory response compared to APOE3/3 mice, but less than in APOE4/4 mice. These data suggest that inhibition of inflammation depends upon the dose of apoE3 protein available and that apoE4 protein may alter inflammation partly by dose effects and partly by being qualitatively different than apoE3 Overall, these data emphasize the important role of apolipoprotein E and of the APOE genotype on the immune responses that are evident in most, in not all, neurological disease.
PMCID: PMC2782461  PMID: 18155324
microglia; apolipoprotein E; innate immunity; cytokine; neuroinflammation
13.  Amyloid reduction by amyloid-β vaccination also reduces mouse tau pathology and protects from neuron loss in two mouse models of Alzheimer’s disease 
Shown to lower amyloid deposits and improve cognition in APP transgenic mouse models, immunotherapy appears to be a promising approach for the treatment of Alzheimer’s disease (AD). Due to limitations in available animal models, however, it has been unclear whether targeting amyloid is sufficient to reduce the other pathological hallmarks of AD—namely, accumulation of pathological, non-mutated tau and neuronal loss. We have now developed two transgenic mouse models (APPSw/NOS2−/− and APPSwDI/NOS2−/−) that more closely model AD. These mice show amyloid pathology, hyperphosphorylated and aggregated normal mouse tau, significant neuron loss, and cognitive deficits. Aβ1–42 or KLH vaccinations were started in these animals at 12 months, when disease progression and cognitive decline are well underway, and continued for 4 months. Vaccinated APPSwDI/NOS2−/− mice, which have predominantly vascular amyloid pathology, showed a 30% decrease in brain Aβ and a 35–45% reduction in hyperphosphorylated tau. Neuron loss and cognitive deficits were partially reduced. In APPSw/NOS2−/− vaccinated mice, brain Aβ was reduced by 65–85% and hyperphosphorylated tau by 50–60 percent. Furthermore, neurons were completely protected, and memory deficits were fully reversed. Microhemorrhage was observed in all vaccinated APPSw/NOS2−/− mice and remains a significant adverse event associated with immunotherapy. Nevertheless, by providing evidence that reducing amyloid pathology also reduces non-mutant tau pathology and blocks neuron loss, these data support the development of amyloid-lowering therapies for disease-modifying treatment of AD.
PMCID: PMC2871319  PMID: 19553436
Immunotherapy; microhemorrhage; neurodegeneration; CAA; amyloid; tau
14.  Vascular amyloid alters astrocytic water and potassium channels in mouse models and humans with Alzheimer’s disease 
Neuroscience  2009;159(3):1055-1069.
The neurovascular unit (NVU) comprises cerebral blood vessels and surrounding astrocytes, neurons, perivascular microglia and pericytes. Astrocytes associated with the NVU are responsible for maintaining cerebral blood flow and ionic and osmotic balances in the brain. A significant proportion of individuals with Alzheimer’s disease (AD) have vascular amyloid deposits (cerebral amyloid angiopathy, CAA) that contribute to the heterogeneous nature of the disease. To determine whether NVU astrocytes are affected by the accumulation of amyloid at cerebral blood vessels we examined astrocytic markers in four transgenic mouse models of amyloid deposition. These mouse models represent mild CAA, moderate CAA with disease progression to tau pathology and neuron loss, severe CAA and severe CAA with disease progression to tau pathology and neuron loss. We found that CAA and disease progression both resulted in distinct NVU astrocytic changes. CAA causes a loss of apparent GFAP-positive astrocytic end-feet and loss of water channels (aquaporin 4) localized to astrocytic end feet. The potassium channels Kir4.1-an inward rectifying potassium channel and BK – a calcium-sensitive large-conductance potassium channel were also lost. The anchoring protein, dystrophin 1, is common to these channels and was reduced in association with CAA. Disease progression was associated with a phenotypic switch in astrocytes indicated by a loss of GFAP-positive cells and a gain of S100β-positive cells. Aquaporin 4, Kir4.1 and dystrophin 1 were also reduced in autopsied brain tissue from individuals with AD that also display moderate and severe CAA. Together, these data suggest that damage to the neurovascular unit may be a factor in the pathogenesis of Alzheimer’s disease.
PMCID: PMC2699894  PMID: 19356689
CAA; astrocytes; amyloid; potassium channels; aquaporin; cerebrovasculature; Alzheimer’s disease
15.  The APOE4 genotype alters the response of microglia and macrophages to 17β-estradiol 
Neurobiology of aging  2007;29(12):1783-1794.
The apolipoprotein E4 (APOE4) gene is a well-known risk factor for Alzheimer’s disease (AD) and other neurological disorders. Post-menopausal women with AD who express at least one APOE4 gene have more severe neuropathology and worsened cognitive scores than their non-expressing counterparts. Since 17β-estradiol down-regulates inflammation as part of its neuroprotective role, we examined the effect of 17β-estradiol on the response of microglia to immune activation as a function of APOE genotype. Our data show that the anti-inflammatory activity of 17β-estradiol is significantly reduced in APOE4 targeted replacement mice compared to APOE3 mice. A significant interaction between APOE genotype and the response to 17β-estradiol was observed for NO and cytokine production by immune activated microglia. The genotype specific effect was not restricted to brain macrophages since peritoneal macrophages from APOE4 ovariectomized mice also demonstrated a significant difference in 17β-estradiol responsiveness. ERβ protein levels in APOE4 microglia were higher than APOE3 microglia, suggesting a difference in post-translational protein regulation in the presence of the APOE4 gene. Overall, our data indicate that the APOE genotype may be a critical component in assessing the effectiveness of 17β-estradiol’s action and may impact the neuroprotective role of 17β-estradiol and of hormone replacement therapy on brain function when the APOE4 gene is expressed.
PMCID: PMC2597534  PMID: 17553597
Inflammation; estrogen; microglia; APOE4; ERα; ERβ; Nitric Oxide; peritoneal macrophage
16.  Heterogeneity of Microglial Activation in the Innate Immune Response in the Brain 
The immune response in the brain has been widely investigated and while many studies have focused on the proinflammatory cytotoxic response, the brain’s innate immune system demonstrates significant heterogeneity. Microglia, like other tissue macrophages, participate in repair and resolution processes after infection or injury to restore normal tissue homeostasis. This review examines the mechanisms that lead to reduction of self-toxicity and to repair and restructuring of the damaged extracellular matrix in the brain. Part of the resolution process involves switching macrophage functional activation to include reduction of proinflammatory mediators, increased production and release of anti-inflammatory cytokines, and production of cytoactive factors involved in repair and reconstruction of the damaged brain. Two partially overlapping and complimentary functional macrophage states have been identified and are called alternative activation and acquired deactivation. The immunosuppressive and repair processes of each of these states and how alternative activation and acquired deactivation participate in chronic neuroinflammation in the brain are discussed.
PMCID: PMC2773116  PMID: 19655259
neuroinflammation; microglia; alternative activation; acquired deactivation; brain repair; immunosuppression; Alzheimer’s disease
17.  The Chemical Biology of Nitric Oxide. Implications in Cellular Signaling 
Nitric oxide (NO) has earned the reputation of being a signaling mediator with many diverse and often opposing biological activities. The diversity in response to this simple diatomic molecule comes from the enormous variety of chemical reactions and biological properties associated with it. In the last few years, the importance of steady state NO concentrations have emerged as a key determinant of its biological function. Precise cellular responses are differentially regulated by specific NO concentration. We propose 5 basic distinct concentration levels of NO activity; cGMP mediated processes ([NO] <1–30 nM; Akt phosphorylation ([NO] = 30–100 nM); stabilization of HIF-1α ([NO] = 100–300 nM); phosphorylation of p53 ([NO] > 400 nM) and nitrosative stress (1 µM). In general, lower NO concentrations promote cell survival and proliferation, while higher levels favor cell cycle arrest, apoptosis, and senescence. Free radical interactions will also influence NO signaling. One of the consequences of reactive oxygen species (ROS) generation is to reduce NO concentrations. This antagonizes the signaling of nitric oxide and in some cases results in converting a cell cycle arrest profile to a cell survival one. The resulting reactive nitrogen species (RNS) that are generated from these reactions can also have biological effects and increase oxidative and nitrosative stress responses. A number of factors determine the formation of NO and its concentration, such as diffusion, consumption, and substrate availability which are referred to as Kinetic Determinants for Molecular Target Interactions. These are the chemical and biochemical parameters that shape cellular responses to NO. Herein we discuss signal transduction and the chemical biology of NO in terms of the direct and indirect reactions.
PMCID: PMC2572721  PMID: 18439435
nitric oxide oxidative nitrosative stress
18.  The Effects of NOS2 Gene Deletion on Mice Expressing Mutated Human AβPP 
Nitric oxide synthase 2 (NOS2) and its gene product, inducible NOS (iNOS) play an important role in neuroinflammation by generating nitric oxide (NO), a critical signaling and redox factor in the brain. Although NO is associated with tissue damage, it can also promote cell survival. We hypothesize that during long-term exposure to amyloid-β (Aβ) in Alzheimer’s disease (AD), NO levels fall in the brain to a threshold at which the protective effects of NO cannot be sustained, promoting Aβ mediated damage. Two new mouse models of AD have been developed that utilize this concept of NO’s action. These mice express human amyloid-β protein precursor (AβPP) mutations that generate Aβ peptides on a mouse NOS2 knockout background. The APP/NOS2−/− bigenic mice progress from Aβ production and amyloid deposition to hyperphosphorylated normal mouse tau at AD-associated epitopes, aggregation and redistribution of tau to somatodendritic regions of neurons and significant neuronal loss including loss of interneurons. This AD-like pathology is accompanied by robust behavioral changes. As APP/NOS2−/− bigenic mice more fully model the human AD disease pathology, they may serve as a tool to better understand disease progression in AD and the role of NO in altering chronic neurological disease processes.
PMCID: PMC2667339  PMID: 19096157
alternative activation; Alzheimer’s disease; amyloid; microglia; mouse models; neuroinflammation; nitric oxide
19.  Immunotherapy, vascular pathology, and microhemorrhages in transgenic mice 
Alzheimer's disease (AD) is a progressive, neurodegenerative disorder that results in severe cognitive decline. Amyloid plaques are a principal pathology found in AD and are composed of aggregated amyloid-beta (Aß) peptides. According to the amyloid hypothesis, Aß peptides initiate the other pathologies characteristic for Alzheimer's disease including cognitive deficits. Immunotherapy against Aß is a potential therapeutic for the treatment of humans with AD. While anti-Aß immunotherapy has been shown to reduce amyloid burden in mouse models and now in humans, immunotherapy also exacerbates vascular pathologies. Cerebral amyloid angiopathy (CAA), that is, the accumulation of amyloid in the cerebrovasculature, is increased with immunotherapy in humans with AD and in mouse models of amyloid deposition. CAA persists in the brains of clinical trial patients that show removal of parenchymal amyloid. Mouse model studies also show that immunotherapy results in multiple small bleeds in the brain, termed microhemorrhages. The neurovascular unit is a term used to describe the cerebrovasculature and its associated cells – astrocytes, neurons, pericytes and microglia. CAA affects brain perfusion and there is now evidence that the neurovascular unit is affected in Alzheimer's disease when CAA is present. Understanding the type of damage to the neurovascular unit caused by CAA in AD and the underlying cause of microhemorrhage after immunotherapy is essential to the success of therapeutic vaccines as a treatment for Alzheimer's disease.
PMCID: PMC2659468  PMID: 19275636
20.  Human Apolipoprotein E Redistributes Fibrillar Amyloid Deposition in Tg-SwDI Mice 
Human apolipoprotein (ApoE) genotype influences the development of Alzheimer’s disease and cerebral amyloid angiopathy (CAA). Specific mutations within the Aβ peptide have been identified that cause familial forms of CAA. However, the effect of APOE genotype on accumulation of CAA mutant Aβ in brain is not well understood. In the present study, we determined how human ApoE3 or ApoE4 influence cerebral Aβ accumulation in transgenic mice (Tg-SwDI) that accumulate human Dutch/Iowa (E22Q/D23N) CAA mutant Aβ in brain, largely in the form of fibrillar cerebral microvascular amyloid. Using Tg-SwDI mice bred onto a human APOE3/3 or human APOE4/4 background, we found that both human ApoE3 and ApoE4 proteins led to a strong reduction in the amount of cerebral microvascular amyloid with an unexpected concomitant appearance of extensive fibrillar parenchymal plaque amyloid. There was strong co-localization of all ApoE proteins with fibrillar amyloid deposits in the mice. In Tg-SwDI/hAPOE3/3 and Tg-SwDI/hAPOE4/4 mice there was no change in the levels of total Aβ40 and Aβ42 or in the amounts of soluble and insoluble Aβ in brain compared to Tg-SwDI mice on the endogenous mouse APOE background. The shift from primarily cerebral microvascular amyloid to parenchymal plaque amyloid in Tg-SwDI/hAPOE3/3 and Tg-SwDI/hAPOE4/4 mice resulted in a parallel shift in the association of activated microglia. These findings indicate that human ApoE has a strong influence on the spatial development of human Dutch/Iowa CAA mutant amyloid accumulation in mouse brain and that microglial activation is in response to the spatial accumulation of fibrillar amyloid.
PMCID: PMC2657728  PMID: 18480287
amyloid β protein; apolipoprotein E, transgenic mice; cerebral vasculature; Alzheimer’s disease; microglia
21.  Progression of amyloid pathology to Alzheimer's pathology in an APP transgenic mouse model by removal of NOS2 
Alzheimer's disease (AD) is characterized by three primary pathologies in the brain; amyloid plaques, neurofibrillary tangles and neuron loss. Mouse models have been useful for studying components of AD but are limited in their ability to fully recapitulate all pathologies. We crossed the APPSwDI transgenic mouse, which develops amyloid ß-(Aß) protein deposits only, with a NOS2 knockout mouse, which develops no AD-like pathology. APPSwDI/NOS2−/− mice displayed impaired spatial memory compared to the APPSwDI mice, yet have the unaltered levels of Aß. APPSwDI mice do not show tau pathology while APPSwDI/NOS2−/− mice displayed extensive tau pathology associated with regions of dense microvascular amyloid deposition. Also, APPSwDI mice do not have any neuron loss while the APPSwDI/NOS2−/− mice have significant neuron loss in the hippocampus and subiculum. Neuropeptide Y neurons have been shown to be particularly vulnerable in AD. These neurons appear to be particularly vulnerable in the APPSwDI/NOS2−/− mice as we observe a dramatic reduction in the number of NPY neurons in the hippocampus and subiculum. These data show that removal of NOS2 from an APP transgenic mouse results in development of full AD-like pathology and behavioral impairments.
PMCID: PMC2621082  PMID: 18272675
amyloid; tau; neuodegeneration; neuropeptide Y; Cerebral amyloid angiopathy
22.  Anti-Aß immunotherapy in Alzheimer's disease; relevance of transgenic mouse studies to clinical trials 
Therapeutic approaches to the treatment of Alzheimer's disease are focused primarily on the Aß peptide which aggregates to form amyloid deposits in the brain. The amyloid hypothesis states that amyloid is the precipitating factor that results in the other pathologies of Alzheimer's, namely neurofibrillary tangles and neurodegeneration, as well as the clinical dementia. One such therapy that has attracted significant attention is anti-Aß immunotherapy. First described in 1999, immunotherapy uses anti-Aß antibodies to lower brain amyloid levels. Active immunization, in which Aß is combined with an adjuvant to stimulate an immune response producing antibodies and passive immunization, in which antibodies are directly injected, were shown to lower brain amyloid levels and improve cognition in multiple transgenic mouse models. Mechanisms of action were studied in these mice and revealed a complex set of mechanisms that depended on the type of antibody used. When active immunization advanced to clinical trials a subset of patients developed meningoencephalitis; an event not predicted in mouse studies. However, it was suspected that a T-cell response due to the type of adjuvant used was the cause of the meningoencephalitis and studies in mice indicated alternative methods of vaccination. Passive immunization has also advanced to phase III clinical trials on the basis of successful transgenic mouse studies. Reports from the active immunization clinical trial indicated that, indeed, amyloid levels in brain were reduced. While APP transgenic mouse models are useful in studying amyloid pathology these mice do not generate significant tau pathology or neuron loss. Continued development of new mouse models that do generate all of these pathologies will be critical in more accurately testing therapeutics and predicting the clinical outcome of such therapeutics.
PMCID: PMC2615484  PMID: 19096156
23.  Quantitative measurement of postural sway in mouse models of human neurodegenerative disease 
Neuroscience  2007;148(4):825-832.
Detection of motor dysfunction in genetic mouse models of neurodegenerative disease requires reproducible, standardized and sensitive behavioral assays. We have utilized a Center of Pressure (CoP) assay in mice to quantify postural sway produced by genetic mutations that affect motor control centers of the brain. As a positive control for postural instability, wild type mice were injected with harmaline, a tremorigenic agent, and the average areas of the 95% confidence ellipse, which measures 95% of the CoP trajectory values recorded in a single trial, were measured. Ellipse area significantly increased in mice treated with increasing doses of harmaline and returned to control values after recovery. We also examined postural sway in mice expressing mutations that mimic frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) (T-279, P301L or P301L-NOS2−/− mice) and that demonstrate motor symptoms. These mice were then compared to a mouse model of Alzheimer’s disease (APPSwDI) that demonstrates cognitive, but not motor deficits. T-279 and P301L-NOS2−/− mice demonstrated a significant increase in CoP ellipse area compared to appropriate wild type control mice or to mice expressing the P301L mutation alone. In contrast, postural instability was significantly reduced in APPSwDI mice that have cognitive deficits but do not have associated motor deficits. The CoP assay provides a simple, sensitive and quantitative tool to detect motor deficits resulting from postural abnormalities in mice and may be useful in understanding the underlying mechanisms of disease.
PMCID: PMC2175386  PMID: 17764851
Tremor; FTDP-17; mouse models; center of pressure assay; motor behavior; postural sway; neurodegeneration
24.  Expression profiles for macrophage alternative activation genes in AD and in mouse models of AD 
Microglia are associated with neuritic plaques in Alzheimer disease (AD) and serve as a primary component of the innate immune response in the brain. Neuritic plaques are fibrous deposits composed of the amyloid beta-peptide fragments (Abeta) of the amyloid precursor protein (APP). Numerous studies have shown that the immune cells in the vicinity of amyloid deposits in AD express mRNA and proteins for pro-inflammatory cytokines, leading to the hypothesis that microglia demonstrate classical (Th-1) immune activation in AD. Nonetheless, the complex role of microglial activation has yet to be fully explored since recent studies show that peripheral macrophages enter an "alternative" activation state.
To study alternative activation of microglia, we used quantitative RT-PCR to identify genes associated with alternative activation in microglia, including arginase I (AGI), mannose receptor (MRC1), found in inflammatory zone 1 (FIZZ1), and chitinase 3-like 3 (YM1).
Our findings confirmed that treatment of microglia with anti-inflammatory cytokines such as IL-4 and IL-13 induces a gene profile typical of alternative activation similar to that previously observed in peripheral macrophages. We then used this gene expression profile to examine two mouse models of AD, the APPsw (Tg-2576) and Tg-SwDI, models for amyloid deposition and for cerebral amyloid angiopathy (CAA) respectively. AGI, MRC1 and YM1 mRNA levels were significantly increased in the Tg-2576 mouse brains compared to age-matched controls while TNFα and NOS2 mRNA levels, genes commonly associated with classical activation, increased or did not change, respectively. Only TNFα mRNA increased in the Tg-SwDI mouse brain. Alternative activation genes were also identified in brain samples from individuals with AD and were compared to age-matched control individuals. In AD brain, mRNAs for TNFα, AGI, MRC1 and the chitinase-3 like 1 and 2 genes (CHI3L1; CHI3L2) were significantly increased while NOS2 and IL-1β mRNAs were unchanged.
Immune cells within the brain display gene profiles that suggest heterogeneous, functional phenotypes that range from a pro-inflammatory, classical activation state to an alternative activation state involved in repair and extracellular matrix remodeling. Our data suggest that innate immune cells in AD may exhibit a hybrid activation state that includes characteristics of classical and alternative activation.
PMCID: PMC1609108  PMID: 17005052
25.  mNos2 Deletion and Human NOS2 Replacement in Alzheimer Disease Models 
Supplemental digital content is available in the text.
Understanding the pathophysiologic mechanisms underlying Alzheimer disease relies on knowledge of disease onset and the sequence of development of brain pathologies. We present a comprehensive analysis of early and progressive changes in a mouse model that demonstrates a full spectrum of characteristic Alzheimer disease–like pathologies. This model demonstrates an altered immune redox state reminiscent of the human disease and capitalizes on data indicating critical differences between human and mouse immune responses, particularly in nitric oxide levels produced by immune activation of the NOS2 gene. Using the APPSwDI+/+/mNos2−/− (CVN-AD) mouse strain, we show a sequence of pathologic events leading to neurodegeneration,which include pathologically hyperphosphorylated tau in the perforant pathway at 6 weeks of age progressing to insoluble tau, early appearance of β-amyloid peptides in perivascular deposits around blood vessels in brain regions known to be vulnerable to Alzheimer disease, and progression to damage and overt loss in select vulnerable neuronal populations in these regions. The role of species differences between hNOS2 and mNos2 was supported by generating mice in which the human NOS2 gene replaced mNos2. When crossed with CVN-AD mice, pathologic characteristics of this new strain (APPSwDI+/−/HuNOS2tg+/+/mNos2−/−) mimicked the pathologic phenotypes found in the CVN-AD strain.
PMCID: PMC4131941  PMID: 25003233
Alzheimer disease; Magnetic resonance spectroscopy; Mouse model; Neuronal loss; NOS2; Tau pathology

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