Maladaptive integrated stress response (ISR) activation is observed in human diseases of the brain. Genetic mutations of eIF2B, a critical mediator of protein synthesis, cause chronic pathway activation resulting in a leukodystrophy, but the precise mechanism is unknown. We generated N208Y eIF2B-α mice and found that this metabolite binding mutation led to destabilization of eIF2B-α, a systemic ISR, and neonatal lethality. 2BAct, an eIF2B activator, rescued lethality and significantly extended the lifespan of this severe model, underscoring its therapeutic potential in pediatric disease. Continuous treatment was required for survival, as withdrawal led to ISR induction in all tissues and rapid deterioration, thereby providing a model to assess the impact of the ISR in vivo by tuning drug availability. Single nuclei RNA-seq of the CNS identified astrocytes, oligodendrocytes, and ependymal cells as the cell types most susceptible to eIF2B dysfunction and revealed dysfunctional maturation of oligodendrocytes. Moreover, ISR activation decreased cholesterol biosynthesis, a process critical for myelin formation and maintenance. As such, persistent ISR engagement may contribute to pathology in other demyelinating diseases.

Provide scientific rationale and clinical approach to test ABBV-CLS-7262 as a novel therapy for Vanishing White Matter disease

eIF2B is a decameric guanine nucleotide exchange factor (GEF) that is essential for protein synthesis and a key effector of the integrated stress response (ISR). Hypomorphic mutations in any of the eIF2B subunits are associated with Vanishing White Matter Disease (VWM), a leukodystrophy characterized by ISR activation and white matter loss. Here, we showed that the VWM-associated N208Y eIF2Bα mutation, which abolishes sugar phosphate binding, led to a drastic reduction in its level in cells and concomitant ISR activation. We found that N208Y homozygous mice are small and die shortly after birth. Remarkably, continuous availability of 2BAct, a small molecule eIF2B activator, in food rescued the lethality and significantly extended their lifespan. 2BAct-maintained N208Y homozygous mice, however, developed motor deficits and loss of myelin with age. As is the case for milder VWM models, ISR induction was restricted to the central nervous system in treated animals. Upon 2BAct withdrawal, adult mutant mice deteriorated quickly, the ISR was induced in all peripheral tissues tested and resulted in high levels of circulating FGF21 and GDF15. This model provides a novel platform to study the impact of ISR activation across tissues with temporal control.

Apolipoprotein E transports lipids and couples metabolism between astrocytes and neurons. The E4 variant (APOE4) affects these functions and represents a genetic predisposition for Alzheimer's disease, but the molecular mechanisms remain elusive. We show that ApoE produces different types of lipoproteins via distinct lipidation pathways. ApoE forms high-density lipoprotein (HDL)-like, cholesterol-rich particles via the ATP binding cassette transporter 1 (ABCA1), a mechanism largely unaffected by ApoE polymorphism. Alternatively, ectopic accumulation of fat in astrocytes, a stress-associated condition, redirects ApoE toward the assembly and secretion of triacylglycerol-rich lipoproteins, a process boosted by the APOE4 variant. We demonstrate in vitro that ApoE can detect triacylglycerol in membranes and spontaneously assemble lipoprotein particles (10-20 nm) rich in unsaturated triacylglycerol, and that APOE4 has remarkable properties behaving as a strong triacylglycerol binder. We propose that fatty APOE4 astrocytes have reduced ability to clear toxic fatty acids from the extracellular milieu, because APOE4 reroutes them back to secretion.

AbstractBackgroundAlzheimer’s Disease (AD) is the most common cause of dementia in the elderly and affects over 35 million people worldwide, imposing increasing social and economic burden as the population ages. While it is widely known that the most prominent genetic risk factor for AD is the presence of the Apolipoprotein E (APOE) ε4 allele, the effects of APOE in the development of AD is still poorly understood. As part of the IMI ADAPTED consortium, we aim to clarify the role of APOE as a risk factor in the development of AD. Here we present an in‐depth analysis of the effect of the APOE genotype on the transcriptome of brain cells derived from human‐induced pluripotent stem cells (hiPSCs).MethodIsogenic hiPSC lines were modified to carry different APOE genotypes: ε3/ε3, ε4/ε4, ε3/ε4, ε2/ε2, as well as an APOE knock‐out (KO) cell line. Lines carrying each of these genotypes were differentiated into distinct cell types. Differential gene expression (DGE) and protein expression (DPE) was calculated, followed by gene set enrichment. Clustering approaches were used to identify shared and differing gene signatures across genotypes. We further applied upstream regulator and network analysis on the individual cell‐type results and integrated these results across cell types. The results were compared with DGE and DEP results from an APOE mouse model. Finally, the identified genes and mechanisms were combined with the results of data from postmortem human brain samples of AD cases and controls.ResultsThe observed transcriptional changes confirmed phenotypic observations made for the hiPSCs and refined the insight of genes identified human brain OMICS data. Several genes and pathways were identified, which showed consistent gene expression on transcriptome and proteome level. Further, shared patterns of expressions of genes across genotypes and potential mechanisms involved in this were detected.ConclusionsIn depth transcriptomics and proteomics analysis of APOE modified hiPSCs enabled to study cell type specific effects and contributed with this to the understanding of mechanisms affected by different APOE genotypes.

Alzheimer's Disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia. The current treatment options for AD are limited to ameliorating cognitive decline temporarily and not reversing or preventing the progression of dementia. Hence, more effective therapeutic strategies are needed to combat this devastating disease. The low-density lipoprotein receptor has been shown to modulate the neuronal metabolism of cholesterol and apolipoprotein E, a major genetic risk factor for AD. LDLR overexpression in mice has been shown to increase amyloid-β clearance and reduce amyloid deposition. We conducted a phenotypic screen to identify novel signaling pathways and targets that regulate LDLR expression in glial cells using an annotated compound library of approximately 29 000 compounds. The screen identified novel targets such as polo like kinase 1 (PLK1), activin receptor like kinase 5 (ALK5), and serotonin transporter (SERT). We used genetic, chemical biology and pathway analysis to confirm the target hypothesis. This work highlights that phenotypic screening is a promising strategy to identify novel mechanisms and targets for therapeutic intervention of complex neurodegenerative disorders.

While being the most prominent genetic risk factor for AD, the effects of APOE variants on biology and the development of AD is still poorly understood. The IMI ADAPTED consortium strives to illuminate the mechanisms underlying the effect of APOE with several approaches. Complementing the reanalysis of the publicly available human data, hiPSC lines with different APOE genotypes (ε3/ε3, ε4/ε4, ε3/ε4, ε2/ε2), differentiated into distinct brain cell types provide a tool to study cell‐type level effects of the APOE genotype. Further, OMICS data of human APOE modified mouse model (ε2/ε2, ε3/ε3, ε4/ε4) allowed cross species comparison and model validation.

AbstractBackgroundOne of the most prominent genetic risk factors for Alzheimer’s Disease (AD) is the Apolipoprotein E (APOE) ε4 allele. Unlike the “neutral” ε3 variant, or the protective ε2variant, the ε4 variant is strongly associated with late‐onset AD, albeit by mechanisms that are not well‐understood.MethodsTo shed light on the biology of APOE in the development of AD, as part of the IMI (Innovative Medicines Initiative) consortium “ADAPTED”, we acquired two human induced pluripotent stem cell (hiPSC) lines, that were isogenically modified to carry the alleles ε2/ε2, ε3/ε3, ε3/ε4, ε4/ε4, as well as a knockout line. Neurons and the phagocytic cells of the CNS, the microglia were differentiated from all ten lines and subjected to thorough phenotyping, including RNA‐seq and proteomics analysis.ResultsCortical neurons do not secrete apoE under normal conditions and treatment of these cells with microglia‐enriched factors and apoE increased their overall Abeta secretion, without significant differences between the various genotypes. Microglia, on the other hand, secrete large amounts of apoE in varying quantities depending on the APOE genotype. Additionally, microglia carrying the ε4/ε4 genotype have a significantly reduced phagocytic capacity compared to their ε3/ε3 counterparts. These observations were supported by whole transcriptome sequencing, which revealed major differences in inflammatory and phagocytic activity‐related gene expression between the different APOE genotypes.ConclusionsThis unique set of ten isogenic hiPSC lines enabled us to assess the cellular phenotypes in functional human neuronal cells. Microglia omics show changes in an array of AD‐relevant pathways, and being the main producers of apoE, these changes need to be understood in depth. Follow‐up experiments will validate candidate genes that might help design new therapies for APOE ε4 AD patients. Disclosure: This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 115975. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA. This study was sponsored by AbbVie Deutschland GmbH & Co. KG. AbbVie contributed to the study design, research, and interpretation of data, writing, reviewing, and approving the abstract. Some authors are currently employed by AbbVie and may own AbbVie stock.

AbstractBackgroundADAPTED (Alzheimer's Disease Apolipoprotein Pathology for Treatment Elucidation and Development) is an Innovative Medicines Initiative (IMI) project that focuses on apolipoprotein E (APOE) as it relates to Alzheimer’s disease. As part of this effort, the utility of APOE transgenic mice as a model of human apoE function was investigated. Specifically, proteomic, metabolomic, and transcriptomic analyses were conducted in aged APOE transgenic mice: ε2/2, ε3/3, ε4/4, knock‐out (KO).MethodsTwo studies were conducted. An initial proteomic and metabolomic study was conducted in brain tissue from male and female 22‐month old APOE ε3/3 and ε4/4 mice. A second proteomic, metabolomic, and transcriptomic study was conducted in brain tissue from 16‐month old APOE ε2/2, ε3/3, ε4/4, or KO mice receiving a low‐fat/low‐sucrose or high‐fat/high‐sucrose diet.ResultsProtein changes (upregulated and downregulated) in 22‐month old APOE ε4 males and females relative to age/sex matched ε3 mice were related to apoE function (LDL), immune function (IgGs) and cellular structure/function. Metabolomic differences in APOE ε4 female and/or male mice relative to ε3 mice were observed for lysophospholipid metabolites, sphinganine; and cholic acid. Enrichment analysis of top genes from the different comparisons involving the ε4 allele (ε 4vs ε3, ε4vs ε2 and ε4 vs KO) showed effects in mitochondrial/respiratory chain and immune‐related pathways.ConclusionProteomic, metabolomic, and transcriptomic examination in APOE ε4 transgenic mice indicate genotype differences consistent with previously described effects of apoE on immune function and lipid signaling. Additional work in ADAPTED will characterize proteomic and metabolomic phenotypes in human‐derived inducible pluripotent stem cells and human patient samples. Comparison of the results across human cells, human samples, and the present findings will demonstrate the potential utility (or lack thereof) for APOE animal models. Disclosure: This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 115975. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA. This study was sponsored by AbbVie, Inc. AbbVie contributed to the study design, research, and interpretation of data, writing, reviewing, and approving the abstract. Some authors are currently employed by AbbVie and may own AbbVie stock.

Apolipoprotein E is a 299-residue lipid carrier protein produced in both the liver and the brain. The protein has three major isoforms denoted apoE2, apoE3, and apoE4 which differ at positions 112 and 158 and which occur at different frequencies in the human population. Genome-wide association studies indicate that the possession of two apoE4 alleles is a strong genetic risk factor for late-onset Alzheimer's disease (LOAD). In an attempt to identify a small molecule stabilizer of apoE4 function that may have utility as a therapy for Alzheimer's disease, we carried out an NMR-based fragment screen on the N-terminal domain of apoE4 and identified a benzyl amidine based fragment binder. In addition to NMR, binding was characterized using various other biophysical techniques, and a crystal structure of the bound core was obtained. Core elaboration ultimately yielded a compound that showed activity in an IL-6 and IL-8 cytokine release assay.

A novel rodent continuous performance test (CPT) was developed as one of the goals of the NEWMEDS (Novel Methods leading to New Medications in Depression and Schizophrenia) consortium to improve its translatability to the CPT test used in human subjects.

The apolipoprotein ε4 allele (apoE4) is a well-established genetic risk factor for late onset Alzheimer's disease (AD), while the apoE2 allele provides protection against AD development. However, the exact molecular mechanisms by which apoE plays its role in AD pathogenesis remain largely unknown. Aiming to investigate the role of apoE in AD, isogenic lines for its different isoforms were generated from four human induced pluripotent stem cell (iPSC) donors within the framework of the IMI2 project ADAPTED. The lines from the first donor were used to derive expandable cortical progenitor cells that can be differentiated into cortical neurons and astrocytes. After characterization of these cells, they were further utilized to study phenotypic differences induced by the various apoE isoforms. It had been previously reported that human apoE might play a role in the inflammatory response of cells. Since inflammation and neurodegeneration are closely linked, we aimed at analyzing the role of apoE in modulating the inflammatory response of isogenic astrocytes. Indeed, stimulating these astrocytes with a cytokine cocktail triggered a differential inflammatory response depending on the apoE genotype. ApoE4 carrying astrocytes displayed stronger pro-inflammatory responses compared to apoE3 cells indicating a potential role for neuro-inflammation in AD. In this study the analysis of these differences was extended to additional apoE genotypes, like apoE2/2 and also included the heterozygote form apoE3/4. ApoE has also been reported to influence the complexity of neuronal networks denoted by the neuron's ability to grow and extend neurites. However, this has not been tested in human-derived cells. Therefore, we investigated the impact of the various apoE isoforms on the differentiation and maturation of cortical neurons under basal conditions and in the presence of various stressors. Since apoE4 is a significant factor in the development of late onset AD, we believe that our mechanistic studies help understand its role in the disease course, and ultimately bring us closer to developing impactful treatments.

Alzheimer's disease (AD) is characterized by memory loss accompanying amyloid beta (Aβ) accumulation, neurofibrillary tangle formation, neuro-inflammation, and neurodegeneration. Despite the development of numerous transgenic (Tg) mouse lines over two decades, an animal model recapitulating a broad set of AD pathology is still needed. Recently, a transgenic rat was developed carrying the familial AD genes Amyloid Precursor Protein (Swedish Mutation) (APPsw) and Presenilin 1 (PS1ΔE9), the TgF344AD rat. These rats are reported to demonstrate age-dependent Aβ accumulation, followed by tauopathy, gliosis, and neuronal loss, as well as cognitive impairment (Cohen et al., 2013; J. Neurosci 33:6245). TgF344AD rats were obtained from the Rat Resource & Research Center (Columbia, MO). AD-like pathology was profiled across several ages (3-16 months), including Aβ plaques measured by 3D6 and mE8 staining, microglial activation measured by Iba1 staining, phosphorylated or aggregated tau as measured by AT8 and AT100, biochemical analysis of Aβ40 and Aβ42, and cognition as measured by a Barnes Maze and a touchscreen Paired Associated Learning (PAL) test. Male and female rats demonstrated an age-dependent increase in Aβ plaques beginning at 6 mo of age in the cortex (CTX) and hippocampus (HPC) and up to at least 15 months of age. Similarly, biochemical analysis of HPC and CTX showed age-dependent increases in Aβ40 and Aβ42 in both males and females. No differences in Iba1 were observed between female Tg and wildtype littermates at 2.5 months of age, but by 12 months of age significant increases were observed in piriform and entorhinal cortices. No differences in AT8 or AT100 in the HPC and overlying CTX were observed in female Tg and wildtype rats at 2.5 or 12 months of age. Modest cognitive impairments were observed in female, but not male, Tg rats tested on a Barnes Maze with a reversal component. No significant differences were observed on Touchscreen PAL. Overall, these data extend earlier findings that show robust amyloid pathology in TgF344AD rats and deficits in a Barnes Maze with a reversal component. Additional analyses are ongoing to determine the extent to which other measures are consistent with previous findings.

ABT-126 is a nicotinic acetylcholine receptor (nAChR) agonist that is selective for the α7 subtype of the receptor. nAChRs are thought to play a role in a variety of neurocognitive processes and have been a pharmacologic target for disorders with cognitive impairment, including schizophrenia and Alzheimer's disease. As part of the preclinical safety package for ABT-126, its potential for abuse was assessed. While the involvement of the α4β2 subtype of the nicotinic receptor in the addictive properties of nicotine has been demonstrated, the role of the α7 receptor has been studied much less extensively. A number of preclinical assays of abuse potential including open-field, drug discrimination and self-administration were employed in male rats. ABT-126 had modest effects on locomotor activity in the open-field assay. In nicotine and d-amphetamine drug discrimination assays, ABT-126 administration failed to produce appreciable d-amphetamine-like or nicotine-like responding, suggesting that its interoceptive effects are distinct from those of these drugs of abuse. In rats trained to self-administer cocaine, substitution with ABT-126 was similar to substitution with saline, indicating that it lacks reinforcing effects. No evidence of physical dependence was noted following subchronic administration. Overall, these data suggest that ABT-126 has a low potential for abuse. Together with other literature on this drug class, it appears that drugs that selectively activate α7 nAChRs are not likely to result in abuse or dependence.

The low rate of success for identifying effective treatments for cognitive dysfunction has prompted recent efforts to improve pharmaceutical discovery and development. In particular, investigators have emphasized improving translation from pre-clinical to clinical research. A specific area of focus has been touchscreen technology; this computer-automated behavioral testing method provides an objective assessment of performance that can be used across species. As part of a larger multi-site study with partners from the Innovative Medicines Initiative (IMI), two US sites, AbbVie and Pfizer, conducted a cross-site experiment with a common protocol for the visual discrimination (VD) task using identical testing equipment, stimuli, and rats of the same strains, sex, and age from the same supplier. As most touchscreen-based rodent experiments have used Lister–Hooded rats that are not readily available outside of Europe, a strain comparison with male Long–Evans rats was conducted as part of the study. Rats were trained for asymptotic performance, and test sessions were performed once per week in a full crossover design with cognition-impairing drugs. Drugs tested were phencyclidine and S-ketamine (N-methyl-d-aspartate (NMDA) antagonists), d-amphetamine (indirect dopamine agonist), and scopolamine (muscarinic antagonist). Satellite brain and plasma samples were taken to confirm appropriate exposures. Results indicate that both rat strains show similar patterns of impairment, although Lister–Hooded rats were more sensitive than Long–Evans rats to three out of four drugs tested. This suggests that researchers should fully explore dose–response relationships in their strain of choice and use care in the interpretation of reversal of cognitive impairment.

Neuronal α4β2* nicotinic acetylcholine receptors mediate cognition, pain, and the discriminative and reinforcing effects of nicotine. In addition to traditional orthosteric agonists, α4β2* positive allosteric modulators (PAMs) have recently been identified. With increased subtype selectivity relative to agonists, PAMs administered alone or in combination with low-dose α4β2* agonists may be used as powerful tools for increasing our understanding of α4β2* pharmacology.

Enhancement of α7 nicotinic receptor (nAChR) activity is considered as an attractive approach for ameliorating cognitive deficits associated with schizophrenia and Alzheimer's disease. Here, we describe the preclinical profile of a novel α7 nAChR agonist, ABT-126. In vitro pharmacology was characterized by radioligand binding, electrophysiology, and Ca2 + imaging methodologies as previously described 1. In vivo studies involved immunohistochemical and behavioral approaches as described 2,3. ABT-126 displayed high affinity to α7 nAChRs (human or rat cortex), but substantially lower affinity at other nAChR subtypes. Functionally, ABT-126 evoked human and rat α7 nAChR current responses in Xenopus oocytes, and enhanced synaptic activity and current responses in rat hippocampal slice preparations. In vivo administration of ABT-126 in rodents modulated biochemical (ERK1/2 and CREB phosphorylation) and neurochemical (ACh release) effects in hippocampal and cortical regions at behaviorally effective dose range. In vivo, ABT-126 was found to be effective in rodent/primate models that capture domains of working memory, memory consolidation and recall, preattention and short-term memory. Repeated daily dosing or steady state exposure of ABT-126 did not result in attenuation of efficacy in vivo. Finally, ABT-126 exhibited acceptable preclinical safety/tolerability profiles. Our studies demonstrate that ABT-126 is a selective α7 nAChR agonist that modulates cortical and hippocampal signaling mechanisms associated with cognitive function. Moreover, ABT-126 exhibited broad-spectrum precognitive efficacy in preclinical models across domains implicated in schizophrenia and Alzheimer's disease. Based on these results, ABT-126 was advanced into clinical development where positive signals of cognitive efficacy were observed [accompanying abstract]. 1 Malysz et al., J. Pharmacol. Exp. Ther., 334: 863, 2010; 2 Bitner et al., J. Neurosci., 27: 10578, 2007; 3 Bitner et al., J. Pharmacol Exp Ther., 334:875, 2010.

Accumulating evidence indicates that schizophrenia and autism spectrum disorder patients are marked by cognitive deficits in working memory and strategy switching. There is accumulating evidence that 5-hydroxytryptamine (5-HT)6 receptors may serve as a useful target to improve cognitive functioning.

Rationale Drug development is a high-risk and high failure enterprise, and studies that provide an early read on the pharmacodynamic activity of novel compounds could save time and money, increasing the efficiency of the drug development process.Objective Preclinical and clinical experiments were designed to examine the utility of the scopolamine-induced cognitive impairment model in predicting pharmacodynamic signals of putatively procognitive compounds, utilizing the acetylcholinesterase inhibitor donepezil for illustration.Methods/Results In normal healthy rats, scopolamine (0.3 mg/kg) significantly impaired performance on the two-platform water maze and on the T-maze. The deficits in water maze performance were reversed by donepezil at 0.5 and 1.0 mg/kg. There was a trend towards reversal of scopolamine-induced deficits in performance on the T-maze with 1.0 mg/kg donepezil. In normal healthy humans, scopolamine (0.3 and 0.5 mg) reliably impaired performance on the Cognitive Drug Research test battery composite scores (power of attention, continuity of attention, quality of working memory, quality of episodic secondary memory, and speed of memory) in a dose-and time-dependent manner. Donepezil (10 mg) significantly attenuated the scopolamine-induced impairment in cognition on power of attention, continuity of attention, quality of working memory, and speed of memory.Conclusions These findings suggest that reversal of scopolamine-induced cognitive impairment is a viable model for predicting pharmacodynamic signals of procognitive compounds in both animals and humans. The utility of the scopolamine-induced cognitive impairment model is discussed and illustrated at various decision points in drug development, with a focus on Go/No Go decisions.