Alzheimer's Disease Protein Relevance Analysis Using Human and Mouse Model Proteomics Data.

The principles governing genotype-phenotype relationships are still emerging(1-3), and detailed translational as well as transcriptomic information is required to understand complex phenotypes, such as the pathogenesis of Alzheimer's disease. For this reason, the proteomics of Alzheimer disease (AD) continues to be studied extensively. Although comparisons between data obtained from humans and mouse models have been reported, approaches that specifically address the between-species statistical comparisons are understudied. Our study investigated the performance of two statistical methods for identification of proteins and biological pathways associated with Alzheimer's disease for cross-species comparisons, taking specific data analysis challenges into account, including collinearity, dimensionality reduction and cross-species protein matching. We used a human dataset from a well-characterized cohort followed for over 22 years with proteomic data available. For the mouse model, we generated proteomic data from whole brains of CVN-AD and matching control mouse models. We used these analyses to determine the reliability of a mouse model to forecast significant proteomic-based pathological changes in the brain that may mimic pathology in human Alzheimer's disease. Compared with LASSO regression, partial least squares discriminant analysis provided better statistical performance for the proteomics analysis. The major biological finding of the study was that extracellular matrix proteins and integrin-related pathways were dysregulated in both the human and mouse data. This approach may help inform the development of mouse models that are more relevant to the study of human late-onset Alzheimer's disease.

Corrigendum: Absolute winding number differentiates mouse spatial navigation strategies with genetic risk for Alzheimer's disease.

[This corrects the article DOI: 10.3389/fnins.2022.848654.].

Infection and inflammation: New perspectives on Alzheimer's disease.

Neuroinflammation has been recognized as a component of Alzheimer's Disease (AD) pathology since the original descriptions by Alois Alzheimer and a role for infections in AD pathogenesis has long been hypothesized. More recently, this hypothesis has gained strength as human genetics and experimental data suggest key roles for inflammatory cells in AD pathogenesis. To review this topic, Duke/University of North Carolina (Duke/UNC) Alzheimer's Disease Research Center hosted a virtual symposium: "Infection and Inflammation: New Perspectives on Alzheimer's Disease (AD)." Participants considered current evidence for and against the hypothesis that AD could be caused or exacerbated by infection or commensal microbes. Discussion focused on connecting microglial transcriptional states to functional states, mouse models that better mimic human immunity, the potential involvement of inflammasome signaling, metabolic alterations, self-reactive T cells, gut microbes and fungal infections, and lessons learned from Covid-19 patients with neurologic symptoms. The content presented in the symposium, and major topics raised in discussions are reviewed in this summary of the proceedings.

Absolute Winding Number Differentiates Mouse Spatial Navigation Strategies With Genetic Risk for Alzheimer's Disease.

Spatial navigation and orientation are emerging as promising markers for altered cognition in prodromal Alzheimer's disease, and even in cognitively normal individuals at risk for Alzheimer's disease. The different APOE gene alleles confer various degrees of risk. The APOE2 allele is considered protective, APOE3 is seen as control, while APOE4 carriage is the major known genetic risk for Alzheimer's disease. We have used mouse models carrying the three humanized APOE alleles and tested them in a spatial memory task in the Morris water maze. We introduce a new metric, the absolute winding number, to characterize the spatial search strategy, through the shape of the swim path. We show that this metric is robust to noise, and works for small group samples. Moreover, the absolute winding number better differentiated APOE3 carriers, through their straighter swim paths relative to both APOE2 and APOE4 genotypes. Finally, this novel metric supported increased vulnerability in APOE4 females. We hypothesized differences in spatial memory and navigation strategies are linked to differences in brain networks, and showed that different genotypes have different reliance on the hippocampal and caudate putamen circuits, pointing to a role for white matter connections. Moreover, differences were most pronounced in females. This departure from a hippocampal centric to a brain network approach may open avenues for identifying regions linked to increased risk for Alzheimer's disease, before overt disease manifestation. Further exploration of novel biomarkers based on spatial navigation strategies may enlarge the windows of opportunity for interventions. The proposed framework will be significant in dissecting vulnerable circuits associated with cognitive changes in prodromal Alzheimer's disease.

Metabolism-Based Gene Differences in Neurons Expressing Hyperphosphorylated AT8- Positive (AT8+) Tau in Alzheimer's Disease.

Metabolic adaptations in the brain are critical to the establishment and maintenance of normal cellular functions and to the pathological responses to disease processes. Here, we have focused on specific metabolic pathways that are involved in immune-mediated neuronal processes in brain using isolated neurons derived from human autopsy brain sections of normal individuals and individuals diagnosed as Alzheimer's disease (AD). Laser capture microscopy was used to select specific cell types in immune-stained thin brain sections followed by NanoString technology to identify and quantify differences in mRNA levels between age-matched control and AD neuronal samples. Comparisons were also made between neurons isolated from AD brain sections expressing pathogenic hyperphosphorylated AT8- positive (AT8+) tau and non-AT8+ AD neurons using double labeling techniques. The mRNA expression data showed unique patterns of metabolic pathway expression between the subtypes of captured neurons that involved membrane based solute transporters, redox factors, and arginine and methionine metabolic pathways. We also identified the expression levels of a novel metabolic gene, Radical-S-Adenosyl Domain1 (RSAD1) and its corresponding protein, Rsad1, that impact methionine usage and radical based reactions. Immunohistochemistry was used to identify specific protein expression levels and their cellular location in NeuN+ and AT8+ neurons. APOE4 vs APOE3 genotype-specific and sex-specific gene expression differences in these metabolic pathways were also observed when comparing neurons from individuals with AD to age-matched individuals.

Capillary Electrophoresis-High Resolution Mass Spectrometry for Measuring In Vivo Arginine Isotope Incorporation in Alzheimer's Disease Mouse Models.

Immune-based metabolic reprogramming of arginine utilization in the brain contributes to the neuronal pathology associated with Alzheimer's disease (AD). To enable our long-term goals of differentiation of AD mouse model genotypes, ages, and sexes based on activity of this pathway, we describe here the novel dosing (using uniformly labeled (13C615N4) arginine) and analysis methods using capillary electrophoresis high-resolution accurate-mass mass spectrometry for isotope tracing of metabolic products of arginine. We developed a pseudoprimed infusion-dosing regimen, using repeated injections, to achieve a steady state of uniformly labeled arginine in 135-195 min post bolus dose. Incorporation of stable isotope labeled carbon and nitrogen from uniformly labeled arginine into a host of downstream metabolites was measured in vivo in mice using serially sampled dried blood spots from the tail. In addition to the dried blood spot time course samples, total isotope incorporation into arginine-related metabolites was measured in the whole brain and plasma after 285 min. Preliminary demonstration of the technique identified differences isotope incorporation in arginine metabolites between male and female mice in a mouse-model of sporadic Alzheimer's disease (APOE4/huNOS2). The technique described herein will permit arginine pathway activity differentiation between mouse genotypes, ages, sexes, or drug treatments in order to elucidate the contribution of this pathway to Alzheimer's disease.

CVN-AD Alzheimer's mice show premature reduction in neurovascular coupling in response to spreading depression and anoxia compared to aged controls.

We compared the efficacy of neurovascular coupling and substrate supply in cerebral cortex during severe metabolic challenges in transgenic Alzheimer's [CVN-AD] and control [C57Bl/6] mice, to evaluate the hypothesis that metabolic insufficiency is a critical component of degeneration leading to dementia. We analyzed cerebral blood flow and metabolic responses to spreading depression (induced by K+ applied to the cortex) and anoxia across aging in CVN-AD + C57Bl/6 genotypes. In the CVN-AD genotype progression to histological and cognitive hallmarks of dementia is a stereotyped function of age. We correlated physiology and imaging of the cortex with the blood flow responses measured with laser doppler probes. The results show that spreading depression resulted in a hyperemic blood flow response that was dramatically reduced (24% in amplitude, 70% in area) in both middle-aged and aged CVN-AD mice compared to C57Bl/6 age-matched controls. However, spreading depression amplitude and conduction velocity (≈6 mm/min) did not differ among groups. Anoxia (100% N2 ) showed significantly decreased (by 62%) reactive blood flow and autoregulation in aged AD-CVN mice compared to aged control animals. Significantly reduced neurovascular coupling occurred prematurely with aging in CVN-AD mice. Abbreviated physiological hyperemia and decreased resilience to anoxia may enhance early-onset metabolic deficiency through decreased substrate supply to the brain. Metabolic deficiency may contribute significantly to the degeneration associated with dementia as a function of aging and regions of the brain involved.

Likelihood ratio statistics for gene set enrichment in Alzheimer's disease pathways.

INTRODUCTION: The study of Alzheimer's disease (AD) has revealed biological pathways with implications for disease neuropathology and pathophysiology. These pathway-level effects may also be mediated by individual characteristics or covariates such as age or sex. Evaluation of AD biological pathways in the context of interactions with these covariates is critical to the understanding of AD as well as the development of model systems used to study the disease. METHODS: Gene set enrichment methods are powerful tools used to interpret gene-level statistics at the level of biological pathways. We introduce a method for quantifying gene set enrichment using likelihood ratio-derived test statistics (gsLRT), which accounts for sample covariates like age and sex. We then use our method to test for age and sex interactions with protein expression levels in AD and to compare the pathway results between human and mouse species. RESULTS: Our method, based on nested logistic regressions is competitive with the existing standard for gene set testing in the context of linear models and complex experimental design. The gene sets we identify as having a significant association with AD-both with and without additional covariate interactions-are validated by previous studies. Differences between gsLRT results on mouse and human datasets are observed. DISCUSSION: Characterizing biological pathways involved in AD builds on the important work involving single gene drivers. Our gene set enrichment method finds pathways that are significantly related to AD while accounting for covariates that may be relevant to disease development. The method highlights commonalities and differences between human AD and mouse models, which may inform the development of higher fidelity models for the study of AD.

Neurovascular and immune mechanisms that regulate postoperative delirium superimposed on dementia.

OBJECTIVE: The present work evaluates the relationship between postoperative immune and neurovascular changes and the pathogenesis of surgery-induced delirium superimposed on dementia. BACKGROUND AND RATIONALE: Postoperative delirium is a common complication in many older adults and in patients with dementia including Alzheimer's disease (AD). The course of delirium can be particularly debilitating, while its pathophysiology remains poorly defined. HISTORICAL EVOLUTION: As of 2019, an estimated 5.8 million people of all ages have been diagnosed with AD, 97% of whom are >65 years of age. Each year, many of these patients require surgery. However, anesthesia and surgery can increase the risk for further cognitive decline. Surgery triggers neuroinflammation both in animal models and in humans, and a failure to resolve this inflammatory state may contribute to perioperative neurocognitive disorders as well as neurodegenerative pathology. UPDATED HYPOTHESIS: We propose an immunovascular hypothesis whereby dysregulated innate immunity negatively affects the blood-brain interface, which triggers delirium and thereby exacerbates AD neuropathology. EARLY EXPERIMENTAL DATA: We have developed a translational model to study delirium superimposed on dementia in APPSwDI/mNos2-/- AD mice (CVN-AD) after orthopedic surgery. At 12 months of age, CVN-AD showed distinct neuroimmune and vascular impairments after surgery, including acute microgliosis and amyloid-ß deposition. These changes correlated with attention deficits, a core feature of delirium-like behavior. FUTURE EXPERIMENTS AND VALIDATION STUDIES: Future research should determine the extent to which prevention of surgery-induced microgliosis and/or neurovascular unit dysfunction can prevent or ameliorate postoperative memory and attention deficits in animal models. Translational human studies should evaluate perioperative indices of innate immunity and neurovascular integrity and assess their potential link to perioperative neurocognitive disorders. MAJOR CHALLENGES FOR THE HYPOTHESIS: Understanding the complex relationships between delirium and dementia will require mechanistic studies aimed at evaluating the role of postoperative neuroinflammation and blood-brain barrier changes in the setting of pre-existing neurodegenerative and/or aging-related pathology. LINKAGE TO OTHER MAJOR THEORIES: Non-resolving inflammation with vascular disease that leads to cognitive impairments and dementia is increasingly important in risk stratification for AD in the aging population. The interdependence of these factors with surgery-induced neuroinflammation and cognitive dysfunction is also becoming apparent, providing a strong platform for assessing the relationship between postoperative delirium and longer term cognitive dysfunction in older adults.

Vascular Cellular Adhesion Molecule-1 (VCAM-1) and Memory Impairment in African-Americans after Small Vessel-Type Stroke.

BACKGROUND: African-Americans (AA) are 3 times more likely to have small-vessel-type ischemic strokes (SVS) than Whites. Small vessel strokes are associated with cognitive impairment, a relationship incompletely explained by white matter hyperintensity (WMH) burden. We examined whether inflammatory/endothelial dysfunction biomarkers are associated with cognition after SVS in AAs. METHODS: Biomarkers were obtained in 24 subjects (median age 56.5 years, 54% women, median 12 years education). Cognition was assessed more than 6 weeks poststroke using the memory composite score (MCS), which was generated using recall from the Hopkins Verbal Learning Test-II and Brief Visuospatial Memory Test-Revised. A semi-automated, volumetric protocol was used to quantify WMH volume (WMHv) on clinical MRI scans. Potential biomarkers including vascular cell adhesion molecule-1 (VCAM-1), interleukin-1 receptor antagonist, interleukin-6, interleukin-8, interleukin-10, interferon gamma, and thrombin-antithrombin (TAT) were log-transformed and correlated with MCS with adjustment for potential confounders. RESULTS: Among serum biomarkers, only VCAM-1-correlated with poorer memory based on the MCS (r = -.659; P = .0006). VCAM-1 (r = .554; P = .005) and age (r = .479; P = .018) correlated with WMHv; VCAM-1 was independently associated with MCS after adjustment for WMHv, age, and education (P = .023). CONCLUSIONS: The findings of this exploratory analysis suggest that endothelial dysfunction and inflammation as reflected by VCAM-1 levels may play a role in poststroke cognitive impairment. Additional studies are needed to validate this observation and to evaluate this relationship in non-AAs and with other stroke types and compare this finding to cognitive impairment in nonstroke populations.

Skyline for Small Molecules: A Unifying Software Package for Quantitative Metabolomics.

Vendor-independent software tools for quantification of small molecules and metabolites are lacking, especially for targeted analysis workflows. Skyline is a freely available, open-source software tool for targeted quantitative mass spectrometry method development and data processing with a 10 year history supporting six major instrument vendors. Designed initially for proteomics analysis, we describe the expansion of Skyline to data for small molecule analysis, including selected reaction monitoring, high-resolution mass spectrometry, and calibrated quantification. This fundamental expansion of Skyline from a peptide-sequence-centric tool to a molecule-centric tool makes it agnostic to the source of the molecule while retaining Skyline features critical for workflows in both peptide and more general biomolecular research. The data visualization and interrogation features already available in Skyline, such as peak picking, chromatographic alignment, and transition selection, have been adapted to support small molecule data, including metabolomics. Herein, we explain the conceptual workflow for small molecule analysis using Skyline, demonstrate Skyline performance benchmarked against a comparable instrument vendor software tool, and present additional real-world applications. Further, we include step-by-step instructions on using Skyline for small molecule quantitative method development and data analysis on data acquired with a variety of mass spectrometers from multiple instrument vendors.

Multivariate MR biomarkers better predict cognitive dysfunction in mouse models of Alzheimer's disease.

To understand multifactorial conditions such as Alzheimer's disease (AD) we need brain signatures that predict the impact of multiple pathologies and their interactions. To help uncover the relationships between pathology affected brain circuits and cognitive markers we have used mouse models that represent, at least in part, the complex interactions altered in AD, while being raised in uniform environments and with known genotype alterations. In particular, we aimed to understand the relationship between vulnerable brain circuits and memory deficits measured in the Morris water maze, and we tested several predictive modeling approaches. We used in vivo manganese enhanced MRI traditional voxel based analyses to reveal regional differences in volume (morphometry), signal intensity (activity), and magnetic susceptibility (iron deposition, demyelination). These regions included hippocampus, olfactory areas, entorhinal cortex and cerebellum, as well as the frontal association area. The properties of these regions, extracted from each of the imaging markers, were used to predict spatial memory. We next used eigenanatomy, which reduces dimensionality to produce sets of regions that explain the variance in the data. For each imaging marker, eigenanatomy revealed networks underpinning a range of cognitive functions including memory, motor function, and associative learning, allowing the detection of associations between context, location, and responses. Finally, the integration of multivariate markers in a supervised sparse canonical correlation approach outperformed single predictor models and had significant correlates to spatial memory. Among a priori selected regions, expected to play a role in memory dysfunction, the fornix also provided good predictors, raising the possibility of investigating how disease propagation within brain networks leads to cognitive deterioration. Our cross-sectional results support that modeling approaches integrating multivariate imaging markers provide sensitive predictors of AD-like behaviors. Such strategies for mapping brain circuits responsible for behaviors may help in the future predict disease progression, or response to interventions.

Identifying Vulnerable Brain Networks in Mouse Models of Genetic Risk Factors for Late Onset Alzheimer's Disease.

The major genetic risk for late onset Alzheimer's disease has been associated with the presence of APOE4 alleles. However, the impact of different APOE alleles on the brain aging trajectory, and how they interact with the brain local environment in a sex specific manner is not entirely clear. We sought to identify vulnerable brain circuits in novel mouse models with homozygous targeted replacement of the mouse ApoE gene with either human APOE3 or APOE4 gene alleles. These genes are expressed in mice that also model the human immune response to age and disease-associated challenges by expressing the human NOS2 gene in place of the mouse mNos2 gene. These mice had impaired learning and memory when assessed with the Morris water maze (MWM) and novel object recognition (NOR) tests. Ex vivo MRI-DTI analyses revealed global and local atrophy, and areas of reduced fractional anisotropy (FA). Using tensor network principal component analyses for structural connectomes, we inferred the pairwise connections which best separate APOE4 from APOE3 carriers. These involved primarily interhemispheric connections among regions of olfactory areas, the hippocampus, and the cerebellum. Our results also suggest that pairwise connections may be subdivided and clustered spatially to reveal local changes on a finer scale. These analyses revealed not just genotype, but also sex specific differences. Identifying vulnerable networks may provide targets for interventions, and a means to stratify patients.

The fornix provides multiple biomarkers to characterize circuit disruption in a mouse model of Alzheimer's disease.

Multivariate biomarkers are needed for detecting Alzheimer's disease (AD), understanding its etiology, and quantifying the effect of therapies. Mouse models provide opportunities to study characteristics of AD in well-controlled environments that can help facilitate development of early interventions. The CVN-AD mouse model replicates multiple AD hallmark pathologies, and we identified multivariate biomarkers characterizing a brain circuit disruption predictive of cognitive decline. In vivo and ex vivo magnetic resonance imaging (MRI) revealed that CVN-AD mice replicate the hippocampal atrophy (6%), characteristic of humans with AD, and also present changes in subcortical areas. The largest effect was in the fornix (23% smaller), which connects the septum, hippocampus, and hypothalamus. In characterizing the fornix with diffusion tensor imaging, fractional anisotropy was most sensitive (20% reduction), followed by radial (15%) and axial diffusivity (2%), in detecting pathological changes. These findings were strengthened by optical microscopy and ultrastructural analyses. Ultrastructual analysis provided estimates of axonal density, diameters, and myelination-through the g-ratio, defined as the ratio between the axonal diameter, and the diameter of the axon plus the myelin sheath. The fornix had reduced axonal density (47% fewer), axonal degeneration (13% larger axons), and abnormal myelination (1.5% smaller g-ratios). CD68 staining showed that white matter pathology could be secondary to neuronal degeneration, or due to direct microglial attack. In conclusion, these findings strengthen the hypothesis that the fornix plays a role in AD, and can be used as a disease biomarker and as a target for therapy.

The effects of the apoE4 genotype on the developing mouse retina.

Apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimer's disease (AD), is associated with neuronal and vascular impairments. The retina, which is as an extension of the central nervous system (CNS), is a particularly suitable model for studying developmental and functional aspects of the neuronal and vascular systems. This study investigates the apoE4-dependent developmental effects on the retinal vasculature and neuronal systems and on the levels of apoE and the vascular endothelial growth factor (VEGF) in the retina. This was performed utilizing retinas of 4, 7, 12, and of 120-day-old human-apoE4-targeted replacement mice and of corresponding mice that express the AD benign isoform, apoE3. The results obtained revealed retinal vascular pathology in the apoE4 mice, which started on the early post-natal days. This includes transient increase in vascular branching, and vascular buds which are round vascular elements representing sprouting or retracting vessels. These effects peaked and ended during the neonatal period. Examination of the synaptic system utilizing the pre-synaptic marker synaptophysin revealed a significant decrease of retinal synaptic density in the apoE4 mice, which was detectable by post-natal day 12 (P12). These morphological changes are associated with neonatal age-dependent elevation in the apoE levels in both apoE3 and apoE4 retinas which is more profound in the apoE4 mice and a corresponding increase in VEGF levels, which is less profound in the apoE4 mice. Additionally, we observed lower levels of retinal VEGF in the apoE4 mice compared to the apoE3 mice retinas on P12. These results show that apoE4 has a transient vascular effect during retinal development that ends in the neonatal period, which is accompanied by a synaptic effect that begins at the end of the neonatal period. These findings show that the apoE4 genotype can have distinct developmental effects on both the retinal vasculature and on neurons and suggest that the vascular effects of apoE4 may be related to reduced levels of VEGF.

Chronic Systemic Immune Dysfunction in African-Americans with Small Vessel-Type Ischemic Stroke.

The incidence of small vessel-type (lacunar) ischemic strokes is greater in African-Americans compared to whites. The chronic inflammatory changes that result from lacunar stroke are poorly understood. To elucidate these changes, we measured serum inflammatory and thrombotic biomarkers in African-Americans at least 6 weeks post-stroke compared to control individuals. Cases were African-Americans with lacunar stroke (n = 30), and controls were age-matched African-Americans with no history of stroke or other major neurologic disease (n = 37). Blood was obtained >6 weeks post-stroke and was analyzed for inflammatory biomarkers. Freshly isolated peripheral blood mononuclear cells were stimulated with lipopolysaccharide (LPS) to assess immune responsiveness in a subset of cases (n = 5) and controls (n = 4). After adjustment for covariates, the pro-inflammatory biomarkers, soluble vascular cadherin adhesion molecule-1 (sVCAM-1) and thrombin anti-thrombin (TAT), were independently associated with lacunar stroke. Immune responsiveness to LPS challenge was abnormal in cases compared to controls. African-Americans with lacunar stroke had elevated blood levels of VCAM-1 and TAT and an abnormal response to acute immune challenge >6 weeks post-stroke, suggesting a chronically compromised systemic inflammatory response.

Arginine deprivation and immune suppression in a mouse model of Alzheimer's disease.

The pathogenesis of Alzheimer's disease (AD) is a critical unsolved question; and although recent studies have demonstrated a strong association between altered brain immune responses and disease progression, the mechanistic cause of neuronal dysfunction and death is unknown. We have previously described the unique CVN-AD mouse model of AD, in which immune-mediated nitric oxide is lowered to mimic human levels, resulting in a mouse model that demonstrates the cardinal features of AD, including amyloid deposition, hyperphosphorylated and aggregated tau, behavioral changes, and age-dependent hippocampal neuronal loss. Using this mouse model, we studied longitudinal changes in brain immunity in relation to neuronal loss and, contrary to the predominant view that AD pathology is driven by proinflammatory factors, we find that the pathology in CVN-AD mice is driven by local immune suppression. Areas of hippocampal neuronal death are associated with the presence of immunosuppressive CD11c(+) microglia and extracellular arginase, resulting in arginine catabolism and reduced levels of total brain arginine. Pharmacologic disruption of the arginine utilization pathway by an inhibitor of arginase and ornithine decarboxylase protected the mice from AD-like pathology and significantly decreased CD11c expression. Our findings strongly implicate local immune-mediated amino acid catabolism as a novel and potentially critical mechanism mediating the age-dependent and regional loss of neurons in humans with AD.

The impact of human and mouse differences in NOS2 gene expression on the brain's redox and immune environment.

BACKGROUND: 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. METHODS/RESULTS: 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. CONCLUSIONS: 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.

mNos2 deletion and human NOS2 replacement in Alzheimer disease models.

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 ß-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(+)/(-)/HuNOS2(tg+)/(+)/mNos2(-/-)) mimicked the pathologic phenotypes found in the CVN-AD strain.