Meeting report of the annual workshop on Principles and Techniques for Improving Preclinical to Clinical Translation in Alzheimer\u27s Disease research.

INTRODUCTION: The second annual 5-day workshop on Principles and Techniques for Improving Preclinical to Clinical Translation in Alzheimer\u27s Disease Research was held October 7-11, 2019, at The Jackson Laboratory in Bar Harbor, Maine, USA, and included didactic lectures and hands-on training. Participants represented a broad range of research across the Alzheimer\u27s disease (AD) field, and varied in career stages from trainees and early stage investigators to established faculty, with attendance from the United States, Europe, and Asia. METHODS: In line with the National Institutes of Health (NIH) initiative on rigor and reproducibility, the workshop aimed to address training gaps in preclinical drug screening by providing participants with the skills and knowledge required to perform pharmacokinetic, pharmacodynamics, and preclinical efficacy experiments. RESULTS: This innovative and comprehensive workshop provided training in fundamental skill sets for executing in vivo preclinical translational studies. DISCUSSION: The success of this workship is expected to translate into practical skills that will enable the goals of improving preclinical to clinical translational studies for AD. HIGHLIGHTS: Nearly all preclinical studies in animal models have failed to translate to successful efficacious medicines for Alzheimer\u27s disease (AD) patients. While a wide variety of potential causes of these failures have been proposed,deficiencies in knowledge and best practices for translational research are not being sufficiently addressed by common training practices. Here we present proceedings from an annual NIA-sponsored workshop focused specifically on preclinical testing paradigms for AD translational research in animal models aimed at enabling improved preclinical to clinical translation for AD

Differential splicing of neuronal genes in a Trem2*R47H mouse model mimics alterations associated with Alzheimer\u27s disease.

BACKGROUND: Molecular characterization of late-onset Alzheimer\u27s disease (LOAD), the leading cause of age-related dementia, has revealed transcripts, proteins, and pathway alterations associated with disease. Assessing these postmortem signatures of LOAD in experimental model systems can further elucidate their relevance to disease origins and progression. Model organisms engineered with human genetic factors further link these signatures to disease-associated variants, especially when studies are designed to leverage homology across species. Here we assess differential gene splicing patterns in aging mouse models carrying humanized APOE4 and/or the Trem2*R47H variant on a C57BL/6J background. We performed a differential expression of gene (DEG) and differential splicing analyses on whole brain transcriptomes at multiple ages. To better understand the difference between differentially expressed and differentially spliced genes, we evaluated enrichment of KEGG pathways and cell-type specific gene signatures of the adult brain from each alteration type. To determine LOAD relevance, we compared differential splicing results from mouse models with multiple human AD splicing studies. RESULTS: We found that differentially expressed genes in Trem2*R47H mice were significantly enriched in multiple AD-related pathways, including immune response, osteoclast differentiation, and metabolism, whereas differentially spliced genes were enriched for neuronal related functions, including GABAergic synapse and glutamatergic synapse. These results were reinforced by the enrichment of microglial genes in DEGs and neuronal genes in differentially spliced genes in Trem2*R47H mice. We observed significant overlap between differentially spliced genes in Trem2*R47H mice and brains from human AD subjects. These effects were absent in APOE4 mice and suppressed in APOE4.Trem2*R47H double mutant mice relative to Trem2*R47H mice. CONCLUSIONS: The cross-species observation that alternative splicing observed in LOAD are present in Trem2*R47H mouse models suggests a novel link between this candidate risk gene and molecular signatures of LOAD in neurons and demonstrates how deep molecular analysis of new genetic models links molecular disease outcomes to a human candidate gene

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

Abstract Introduction: Apolipoprotein E (APOE) ε4 is the strongest genetic risk factor for Alzheimer\u27s 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 APOEε3/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\u27s 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 \u27hormone regulation\u27 and \u27insulin signaling,\u27 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 \u27extracellular matrix\u27 and \u27blood coagulation\u27 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

Improving preclinical to clinical translation in Alzheimer\u27s disease research.

Introduction: Preclinical testing in animal models is a critical component of the drug discovery and development process. While hundreds of interventions have demonstrated preclinical efficacy for ameliorating cognitive impairments in animal models, none have confirmed efficacy in Alzheimer\u27s disease (AD) clinical trials. Critically this lack of translation to the clinic points in part to issues with the animal models, the preclinical assays used, and lack of scientific rigor and reproducibility during execution. In an effort to improve this translation, the Preclinical Testing Core (PTC) of the Model Organism Development and Evaluation for Late-onset AD (MODEL-AD) consortium has established a rigorous screening strategy with go/no-go decision points that permits unbiased assessments of therapeutic agents. Methods: An initial screen evaluates drug stability, formulation, and pharmacokinetics (PK) to confirm appreciable brain exposure in the disease model at the pathologically relevant ages, followed by pharmacodynamics (PD) and predictive PK/PD modeling to inform the dose regimen for long-term studies. The secondary screen evaluates target engagement and disease modifying activity using non-invasive positron emission tomography/magnetic resonance imaging (PET/MRI). Provided the compound meets its go criteria for these endpoints, evaluation for efficacy on behavioral endpoints are conducted. Results: Validation of this pipeline using tool compounds revealed the importance of critical quality control (QC) steps that researchers need to be aware of when executing preclinical studies. These include confirmation of the active pharmaceutical ingredient and at the precise concentration expected; and an experimental design that is well powered and in line with the Animal Research Reporting of In vivo Experiments (ARRIVE) guidelines. Discussion: Taken together our experience executing a rigorous screening strategy with QC checkpoints provides insight to the challenges of conducting translational studies in animal models. The PTC pipeline is a National Institute on Aging (NIA)-supported resource accessible to the research community for investigators to nominate compounds for testing (https://stopadportal.synapse.org/), and these resources will ultimately enable better translational studies to be conducted

STOP-AD portal: Selecting the optimal pharmaceutical for preclinical drug testing in Alzheimer\u27s disease.

We propose an unbiased methodology to rank compounds for advancement into comprehensive preclinical testing for Alzheimer\u27s disease (AD). Translation of compounds to the clinic in AD has been hampered by poor predictive validity of models, compounds with limited pharmaceutical properties, and studies that lack rigor. To overcome this, MODEL-AD\u27s Preclinical Testing Core developed a standardized pipeline for assessing efficacy in AD mouse models. We hypothesize that rank-ordering compounds based upon pharmacokinetic, efficacy, and toxicity properties in preclinical models will enhance successful translation to the clinic. Previously compound selection was based solely on physiochemical properties, with arbitrary cutoff limits, making ranking challenging. Since no gold standard exists for systematic prioritization, validating a selection criteria has remained elusive. The STOP-AD framework evaluates the drug-like properties to rank compounds for in vivo studies, and uses an unbiased approach that overcomes the validation limitation by performing Monte-Carlo simulations. HIGHLIGHTS: Promising preclinical studies for AD drugs have not translated to clinical success. Systematic assessment of AD drug candidates may increase clinical translatability. We describe a well-defined framework for compound selection with clear selection metrics

Perivascular cells induce microglial phagocytic states and synaptic engulfment via SPP1 in mouse models of Alzheimer's disease

Alzheimer's disease (AD) is characterized by synaptic loss, which can result from dysfunctional microglial phagocytosis and complement activation. However, what signals drive aberrant microglia-mediated engulfment of synapses in AD is unclear. Here we report that secreted phosphoprotein 1 (SPP1/osteopontin) is upregulated predominantly by perivascular macrophages and, to a lesser extent, by perivascular fibroblasts. Perivascular SPP1 is required for microglia to engulf synapses and upregulate phagocytic markers including C1qa, Grn and Ctsb in presence of amyloid-β oligomers. Absence of Spp1 expression in AD mouse models results in prevention of synaptic loss. Furthermore, single-cell RNA sequencing and putative cell-cell interaction analyses reveal that perivascular SPP1 induces microglial phagocytic states in the hippocampus of a mouse model of AD. Altogether, we suggest a functional role for SPP1 in perivascular cells-to-microglia crosstalk, whereby SPP1 modulates microglia-mediated synaptic engulfment in mouse models of AD

Perivascular cells induce microglial phagocytic states and synaptic engulfment via SPP1 in mouse models of Alzheimer\u27s disease.

Alzheimer\u27s disease (AD) is characterized by synaptic loss, which can result from dysfunctional microglial phagocytosis and complement activation. However, what signals drive aberrant microglia-mediated engulfment of synapses in AD is unclear. Here we report that secreted phosphoprotein 1 (SPP1/osteopontin) is upregulated predominantly by perivascular macrophages and, to a lesser extent, by perivascular fibroblasts. Perivascular SPP1 is required for microglia to engulf synapses and upregulate phagocytic markers including C1qa, Grn and Ctsb in presence of amyloid-β oligomers. Absence of Spp1 expression in AD mouse models results in prevention of synaptic loss. Furthermore, single-cell RNA sequencing and putative cell-cell interaction analyses reveal that perivascular SPP1 induces microglial phagocytic states in the hippocampus of a mouse model of AD. Altogether, we suggest a functional role for SPP1 in perivascular cells-to-microglia crosstalk, whereby SPP1 modulates microglia-mediated synaptic engulfment in mouse models of AD

A novel systems biology approach to evaluate mouse models of late-onset Alzheimer\u27s disease.

BACKGROUND: Late-onset Alzheimer\u27s disease (LOAD) is the most common form of dementia worldwide. To date, animal models of Alzheimer\u27s have focused on rare familial mutations, due to a lack of frank neuropathology from models based on common disease genes. Recent multi-cohort studies of postmortem human brain transcriptomes have identified a set of 30 gene co-expression modules associated with LOAD, providing a molecular catalog of relevant endophenotypes. RESULTS: This resource enables precise gene-based alignment between new animal models and human molecular signatures of disease. Here, we describe a new resource to efficiently screen mouse models for LOAD relevance. A new NanoString nCounter® Mouse AD panel was designed to correlate key human disease processes and pathways with mRNA from mouse brains. Analysis of the 5xFAD mouse, a widely used amyloid pathology model, and three mouse models based on LOAD genetics carrying APOE4 and TREM2*R47H alleles demonstrated overlaps with distinct human AD modules that, in turn, were functionally enriched in key disease-associated pathways. Comprehensive comparison with full transcriptome data from same-sample RNA-Seq showed strong correlation between gene expression changes independent of experimental platform. CONCLUSIONS: Taken together, we show that the nCounter Mouse AD panel offers a rapid, cost-effective and highly reproducible approach to assess disease relevance of potential LOAD mouse models

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

Late-onset Alzheimer\u27s 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