Assessment of neurovascular uncoupling: APOE status is a key driver of early metabolic and vascular dysfunction.

BACKGROUND: Alzheimer's disease (AD) is the most common cause of dementia worldwide, with apolipoprotein Eε4 (APOEε4) being the strongest genetic risk factor. Current clinical diagnostic imaging focuses on amyloid and tau; however, new methods are needed for earlier detection. METHODS: PET imaging was used to assess metabolism-perfusion in both sexes of aging C57BL/6J, and hAPOE mice, and were verified by transcriptomics, and immunopathology. RESULTS: All hAPOE strains showed AD phenotype progression by 8 months, with females exhibiting the regional changes, which correlated with GO-term enrichments for glucose metabolism, perfusion, and immunity. Uncoupling analysis revealed APOEε4/ε4 exhibited significant Type-1 uncoupling (↓ glucose uptake, ↑ perfusion) at 8 and 12 months, while APOEε3/ε4 demonstrated Type-2 uncoupling (↑ glucose uptake, ↓ perfusion), while immunopathology confirmed cell specific contributions. DISCUSSION: This work highlights APOEε4 status in AD progression manifests as neurovascular uncoupling driven by immunological activation, and may serve as an early diagnostic biomarker. HIGHLIGHTS: We developed a novel analytical method to analyze PET imaging of 18F-FDG and 64Cu-PTSM data in both sexes of aging C57BL/6J, and hAPOEε3/ε3, hAPOEε4/ε4, and hAPOEε3/ε4 mice to assess metabolism-perfusion profiles termed neurovascular uncoupling. This analysis revealed APOEε4/ε4 exhibited significant Type-1 uncoupling (decreased glucose uptake, increased perfusion) at 8 and 12 months, while APOEε3/ε4 demonstrated significant Type-2 uncoupling (increased glucose uptake, decreased perfusion) by 8 months which aligns with immunopathology and transcriptomic signatures. This work highlights that there may be different mechanisms underlying age related changes in APOEε4/ε4 compared with APOEε3/ε4. We predict that these changes may be driven by immunological activation and response, and may serve as an early diagnostic biomarker.

Three major effects of APOEε4 on Aß immunotherapy induced ARIA.

The targeting of amyloid-beta (Aß) plaques therapeutically as one of the primary causes of Alzheimer's disease (AD) dementia has been an ongoing effort spanning decades. While some antibodies are extremely promising and have been moved out of clinical trials and into the clinic, most of these treatments show similar adverse effects in the form of cerebrovascular damage known as amyloid-related imaging abnormalities (ARIA). The two categories of ARIA are of major concern for patients, families, and prescribing physicians, with ARIA-E presenting as cerebral edema, and ARIA-H as cerebral hemorrhages (micro- and macro-). From preclinical and clinical trials, it has been observed that the greatest genetic risk factor for AD, APOEε4, is also a major risk factor for anti-Aß immunotherapy-induced ARIA. APOEε4 carriers represent a large population of AD patients, and, therefore, limits the broad adoption of these therapies across the AD population. In this review we detail three hypothesized mechanisms by which APOEε4 influences ARIA risk: (1) reduced cerebrovascular integrity, (2) increased neuroinflammation and immune dysregulation, and (3) elevated levels of CAA. The effects of APOEε4 on ARIA risk is clear, however, the underlying mechanisms require more research.

Soluble Biomarkers of Cerebrovascular Pathologies.

Vascular contributions to cognitive impairment and dementia (VCID) is an all-encompassing term that describes cognitive impairment due to cerebrovascular origins. With the advancement of imaging and pathological studies, we now understand that VCID is often comorbid with Alzheimer disease. While researchers in the Alzheimer disease field have been working for years to establish and test blood-based biomarkers for Alzheimer disease diagnosis, prognosis, clinical therapy discovery, and early detection, blood-based biomarkers for VCID are in their infancy and also face challenges. VCID is heterogeneous, comprising many different pathological entities (ischemic, or hemorrhagic), and spatial and temporal differences (acute or chronic). This review highlights pathways that are aiding the search for sensitive and specific blood-based cerebrovascular dysfunction markers, describes promising candidates, and explains ongoing initiatives to discover blood-based VCID biomarkers.

Assessment of Neurovascular Uncoupling: APOE Status is a Key Driver of Early Metabolic and Vascular Dysfunction.

BACKGROUND: Alzheimer's disease (AD) is the most common cause of dementia worldwide, with apolipoprotein ε4 (APOEε4) being the strongest genetic risk factor. Current clinical diagnostic imaging focuses on amyloid and tau; however, new methods are needed for earlier detection. METHODS: PET imaging was used to assess metabolism-perfusion in both sexes of aging C57BL/6J, and hAPOE mice, and were verified by transcriptomics, and immunopathology. RESULTS: All hAPOE strains showed AD phenotype progression by 8 mo, with females exhibiting the regional changes, which correlated with GO-term enrichments for glucose metabolism, perfusion, and immunity. Uncoupling analysis revealed APOEε4/ε4 exhibited significant Type-1 uncoupling (↓ glucose uptake, ↑ perfusion) at 8 and 12 mo, while APOEε3/ε4 demonstrated Type-2 uncoupling (↑ glucose uptake, ↓ perfusion), while immunopathology confirmed cell specific contributions. DISCUSSION: This work highlights APOEε4 status in AD progression manifest as neurovascular uncoupling driven by immunological activation, and may serve as an early diagnostic biomarker.

Alzheimer's disease and inflammatory biomarkers positively correlate in plasma in the UK-ADRC cohort.

INTRODUCTION: Protein-based plasma assays provide hope for improving accessibility and specificity of molecular diagnostics to diagnose dementia. METHODS: Plasma was obtained from participants (N = 837) in our community-based University of Kentucky Alzheimer's Disease Research Center cohort. We evaluated six Alzheimer's disease (AD)- and neurodegeneration-related (Aß40, Aß42, Aß42/40, p-tau181, total tau, and NfLight) and five inflammatory biomarkers (TNF𝛼, IL6, IL8, IL10, and GFAP) using the SIMOA-based protein assay platform. Statistics were performed to assess correlations. RESULTS: Our large cohort reflects previous plasma biomarker findings. Relationships between biomarkers to understand AD-inflammatory biomarker correlations showed significant associations between AD and inflammatory biomarkers suggesting peripheral inflammatory interactions with increasing AD pathology. Biomarker associations parsed out by clinical diagnosis (normal, MCI, and dementia) reveal changes in strength of the correlations across the cognitive continuum. DISCUSSION: Unique AD-inflammatory biomarker correlations in a community-based cohort reveal a new avenue for utilizing plasma-based biomarkers in the assessment of AD and related dementias. HIGHLIGHTS: Large community cohorts studying sex, age, and APOE genotype effects on biomarkers are few. It is unknown how biomarker-biomarker associations vary through aging and dementia. Six AD (Aß40, Aß42, Aß42/40, p-tau181, total tau, and NfLight) and five inflammatory biomarkers (TNFα, IL6, IL8, IL10, and GFAP) were used to examine associations between biomarkers. Plasma biomarkers suggesting increasing cerebral AD pathology corresponded to increases in peripheral inflammatory markers, both pro-inflammatory and anti-inflammatory. Strength of correlations, between pairs of classic AD and inflammatory plasma biomarker, changes throughout cognitive progression to dementia.

Vascular Considerations for Amyloid Immunotherapy.

PURPOSE OF REVIEW: Amyloid beta (Aß) plaque accumulation is a hallmark pathology contributing to Alzheimer's disease (AD) and is widely hypothesized to lead to cognitive decline. Decades of research into anti-Aß immunotherapies provide evidence for increased Aß clearance from the brain; however, this is frequently accompanied by complicated vascular deficits. This article reviews the history of anti-Aß immunotherapies and clinical findings and provides recommendations moving forward. RECENT FINDINGS: In 20 years of both animal and human studies, anti-Aß immunotherapies have been a prevalent avenue of reducing hallmark Aß plaques. In both models and with different anti-Aß antibody designs, amyloid-related imaging abnormalities (ARIA) indicating severe cerebrovascular compromise have been common and concerning occurrence. ARIA caused by anti-Aß immunotherapy has been noted since the early 2000s, and the mechanisms driving it are still unknown. Recent approval of aducanumab comes with renewed urgency to consider vascular deficits caused by anti-Aß immunotherapy.

APOE ε4 and exercise interact in a sex-specific manner to modulate dementia risk factors.

Introduction: Apolipoprotein E (APOE) ε4 is the strongest genetic risk factor for Alzheimer's disease and related dementias (ADRDs), affecting many different pathways that lead to cognitive decline. Exercise is one of the most widely proposed prevention and intervention strategies to mitigate risk and symptomology of ADRDs. Importantly, exercise and APOE ε4 affect similar processes in the body and brain. While both APOE ε4 and exercise have been studied extensively, their interactive effects are not well understood. Methods: To address this, male and female APOE ε3/ε3, APOE ε3/ε4, and APOE ε4/ε4 mice ran voluntarily from wean (1 month) to midlife (12 months). Longitudinal and cross-sectional phenotyping were performed on the periphery and the brain, assessing markers of risk for dementia such as weight, body composition, circulating cholesterol composition, murine daily activities, energy expenditure, and cortical and hippocampal transcriptional profiling. Results: Data revealed chronic running decreased age-dependent weight gain, lean and fat mass, and serum low-density lipoprotein concentration dependent on APOE genotype. Additionally, murine daily activities and energy expenditure were significantly influenced by an interaction between APOE genotype and running in both sexes. Transcriptional profiling of the cortex and hippocampus predicted that APOE genotype and running interact to affect numerous biological processes including vascular integrity, synaptic/neuronal health, cell motility, and mitochondrial metabolism, in a sex-specific manner. Discussion: These data in humanized mouse models provide compelling evidence that APOE genotype should be considered for population-based strategies that incorporate exercise to prevent ADRDs and other APOE-relevant diseases.

The APOE ε3/ε4 Genotype Drives Distinct Gene Signatures in the Cortex of Young Mice.

Introduction: Restrictions on existing APOE mouse models have impacted research toward understanding the strongest genetic risk factor contributing to Alzheimer's disease (AD) and dementia, APOEε4 , by hindering observation of a key, common genotype in humans - APOEε3/ε4 . Human studies are typically underpowered to address APOEε4 allele risk as the APOEε4/ε4 genotype is rare, which leaves human and mouse research unsupported to evaluate the APOEε3/ε4 genotype on molecular and pathological risk for AD and dementia. Methods: As a part of MODEL-AD, we created and validated new versions of humanized APOEε3/ε3 and APOEε4/ε4 mouse strains that, due to unrestricted breeding, allow for the evaluation of the APOEε3/ε4 genotype. As biometric measures are often translatable between mouse and human, we profiled circulating lipid concentrations. We also performed transcriptional profiling of the cerebral cortex at 2 and 4 months (mos), comparing APOEε3/ε4 and APOEε4/ε4 to the reference APOEε3/ε3 using linear modeling and WGCNA. Further, APOE mice were exercised and compared to litter-matched sedentary controls, to evaluate the interaction between APOEε4 and exercise at a young age. Results: Expression of human APOE isoforms were confirmed in APOEε3/ε3, APOEε3/ε4 and APOEε4/ε4 mouse brains. At two mos, cholesterol composition was influenced by sex, but not APOE genotype. Results show that the APOEε3/ε4 and APOEε4/ε4 genotype exert differential effects on cortical gene expression. APOEε3/ε4 uniquely impacts 'hormone regulation' and 'insulin signaling,' terms absent in APOEε4/ε4 data. At four mos, cholesterol and triglyceride levels were affected by sex and activity, with only triglyceride levels influenced by APOE genotype. Linear modeling revealed APOEε3/ε4 , but not APOEε4/ε4 , affected 'extracellular matrix' and 'blood coagulation' related terms. We confirmed these results using WGCNA, indicating robust, yet subtle, transcriptional patterns. While there was little evidence of APOE genotype by exercise interaction on the cortical transcriptome at this young age, running was predicted to affect myelination and gliogenesis, independent of APOE genotype with few APOE genotype-specific affects identified. Discussion: APOEε4 allele dosage-specific effects were observed in circulating lipid levels and cortical transcriptional profiles. Future studies are needed to establish how these data may contribute to therapeutic development in APOEε3/ε4 and APOEε4/ε4 dementia patients.

Corrigendum: Uncovering Disease Mechanisms in a Novel Mouse Model Expressing Humanized APOEε4 and Trem2*R47H.

[This corrects the article DOI: 10.3389/fnagi.2021.735524.].

Uncovering Disease Mechanisms in a Novel Mouse Model Expressing Humanized APOEε4 and Trem2*R47H.

Late-onset Alzheimer's disease (AD; LOAD) is the most common human neurodegenerative disease, however, the availability and efficacy of disease-modifying interventions is severely lacking. Despite exceptional efforts to understand disease progression via legacy amyloidogenic transgene mouse models, focus on disease translation with innovative mouse strains that better model the complexity of human AD is required to accelerate the development of future treatment modalities. LOAD within the human population is a polygenic and environmentally influenced disease with many risk factors acting in concert to produce disease processes parallel to those often muted by the early and aggressive aggregate formation in popular mouse strains. In addition to extracellular deposits of amyloid plaques and inclusions of the microtubule-associated protein tau, AD is also defined by synaptic/neuronal loss, vascular deficits, and neuroinflammation. These underlying processes need to be better defined, how the disease progresses with age, and compared to human-relevant outcomes. To create more translatable mouse models, MODEL-AD (Model Organism Development and Evaluation for Late-onset AD) groups are identifying and integrating disease-relevant, humanized gene sequences from public databases beginning with APOEε4 and Trem2*R47H, two of the most powerful risk factors present in human LOAD populations. Mice expressing endogenous, humanized APOEε4 and Trem2*R47H gene sequences were extensively aged and assayed using a multi-disciplined phenotyping approach associated with and relative to human AD pathology. Robust analytical pipelines measured behavioral, transcriptomic, metabolic, and neuropathological phenotypes in cross-sectional cohorts for progression of disease hallmarks at all life stages. In vivo PET/MRI neuroimaging revealed regional alterations in glycolytic metabolism and vascular perfusion. Transcriptional profiling by RNA-Seq of brain hemispheres identified sex and age as the main sources of variation between genotypes including age-specific enrichment of AD-related processes. Similarly, age was the strongest determinant of behavioral change. In the absence of mouse amyloid plaque formation, many of the hallmarks of AD were not observed in this strain. However, as a sensitized baseline model with many additional alleles and environmental modifications already appended, the dataset from this initial MODEL-AD strain serves an important role in establishing the individual effects and interaction between two strong genetic risk factors for LOAD in a mouse host.

Genetic basis of susceptibility to low-dose paraquat and variation between the sexes in Drosophila melanogaster.

Toxicant resistance is a complex trait, affected both by genetics and the environment. Like most complex traits, it can exhibit sexual dimorphism, yet sex is often overlooked as a factor in studies of toxicant resistance. Paraquat, one such toxicant, is a commonly used herbicide and is known to produce mitochondrial oxidative stress, decrease dopaminergic neurons and dopamine (DA) levels, and decrease motor ability. While the main effects of paraquat are well-characterized, less is known about the naturally occurring variation in paraquat susceptibility. The purpose of this study was to map the genes contributing to low-dose paraquat susceptibility in Drosophila melanogaster, and to determine if susceptibility differs between the sexes. One hundred of the Drosophila Genetic Reference Panel (DGRP) lines were scored for susceptibility via climbing ability and used in a genome-wide association study (GWAS). Variation in seventeen genes in females and thirty-five genes in males associated with paraquat susceptibility. Only two candidate genes overlapped between the sexes despite a significant positive correlation between male and female susceptibilities. Many associated polymorphisms had significant interactions with sex, with most having conditionally neutral effects. Conditional neutrality between the sexes probably stems from sex-biased expression which may result from partial resolution of sexual conflict. Candidate genes were verified with RNAi knockdowns, gene expression analyses, and DA quantification. Several of these genes are novel associations with paraquat susceptibility. This research highlights the importance of assessing both sexes when studying toxicant susceptibility.

A novel mouse model expressing human forms for complement receptors CR1 and CR2.

BACKGROUND: The complement cascade is increasingly implicated in development of a variety of diseases with strong immune contributions such as Alzheimer's disease and Systemic Lupus Erythematosus. Mouse models have been used to determine function of central components of the complement cascade such as C1q and C3. However, species differences in their gene structures mean that mice do not adequately replicate human complement regulators, including CR1 and CR2. Genetic variation in CR1 and CR2 have been implicated in modifying disease states but the mechanisms are not known. RESULTS: To decipher the roles of human CR1 and CR2 in health and disease, we engineered C57BL/6J (B6) mice to replace endogenous murine Cr2 with human complement receptors, CR1 and CR2 (B6.CR2CR1). CR1 has an array of allotypes in human populations and using traditional recombination methods (Flp-frt and Cre-loxP) two of the most common alleles (referred to here as CR1long and CR1short) can be replicated within this mouse model, along with a CR1 knockout allele (CR1KO). Transcriptional profiling of spleens and brains identified genes and pathways differentially expressed between mice homozygous for either CR1long, CR1short or CR1KO. Gene set enrichment analysis predicts hematopoietic cell number and cell infiltration are modulated by CR1long, but not CR1short or CR1KO. CONCLUSION: The B6.CR2CR1 mouse model provides a novel tool for determining the relationship between human-relevant CR1 alleles and disease.

Transcriptional profiling predicts running promotes cerebrovascular remodeling in young but not midlife mice.

BACKGROUND: The incidence of dementia and cognitive decline is increasing with no therapy or cure. One of the reasons treatment remains elusive is because there are various pathologies that contribute to age-related cognitive decline. Specifically, with Alzheimer's disease, targeting to reduce amyloid beta plaques and phosphorylated tau aggregates in clinical trials has not yielded results to slow symptomology, suggesting a new approach is needed. Interestingly, exercise has been proposed as a potential therapeutic intervention to improve brain health and reduce the risk for dementia, however the benefits throughout aging are not well understood. RESULTS: To better understand the effects of exercise, we preformed transcriptional profiling on young (1-2 months) and midlife (12 months) C57BL/6 J (B6) male mice after 12 weeks of voluntary running. Data was compared to age-matched sedentary controls. Interestingly, the midlife running group naturally broke into two cohorts based on distance ran - either running a lot and more intensely (high runners) or running less and less intensely (low runners). Midlife high runners had lower LDL cholesterol as well as lower adiposity (%fat) compared to sedentary, than midlife low runners compared to sedentary suggesting more intense running lowered systemic markers of risk for age-related diseases including dementias. Differential gene analysis of transcriptional profiles generated from the cortex and hippocampus showed thousands of differentially expressed (DE) genes when comparing young runners to sedentary controls. However, only a few hundred genes were DE comparing either midlife high runners or midlife low runners to midlife sedentary controls. This indicates that, in our study, the effects of running are reduced through aging. Gene set enrichment analyses identified enrichment of genes involved in extracellular matrix (ECM), vascular remodeling and angiogenesis in young runners but not midlife runners. These genes are known to be expressed in multiple vascular-related cell types including astrocytes, endothelial cells, pericytes and smooth muscle cells. CONCLUSIONS: Taken together these results suggest running may best serve as a preventative measure to reduce risk for cerebrovascular decline. Ultimately, this work shows that exercise may be more effective to prevent dementia if introduced at younger ages.

Meox2 Haploinsufficiency Accelerates Axonal Degeneration in DBA/2J Glaucoma.

Purpose: Glaucoma is a complex disease with major risk factors including advancing age and increased intraocular pressure (IOP). Dissecting these earliest events will likely identify new avenues for therapeutics. Previously, we performed transcriptional profiling in DBA/2J (D2) mice, a widely used mouse model relevant to glaucoma. Here, we use these data to identify and test regulators of early gene expression changes in DBA/2J glaucoma. Methods: Upstream regulator analysis (URA) in Ingenuity Pathway Analysis was performed to identify potential master regulators of differentially expressed genes. The function of one putative regulator, mesenchyme homeobox 2 (Meox2), was tested using a combination of genetic, biochemical, and immunofluorescence approaches. Results: URA identified Meox2 as a potential regulator of early gene expression changes in the optic nerve head (ONH) of DBA/2J mice. Meox2 haploinsufficiency did not affect the characteristic diseases of the iris or IOP elevation seen in DBA/2J mice but did cause a significant increase in the numbers of eyes with axon damage compared to controls. While young mice appeared normal, aged Meox2 haploinsufficient DBA/2J mice showed a 44% reduction in MEOX2 protein levels. This correlated with modulation of age- and disease-specific vascular and myeloid alterations. Conclusions: Our data support a model whereby Meox2 controls IOP-dependent vascular remodeling and neuroinflammation to promote axon survival. Promoting these earliest responses prior to IOP elevation may be a viable neuroprotective strategy to delay or prevent human glaucoma.