Variant-to-function mapping of late-onset Alzheimer's disease GWAS signals in human microglial cell models implicates RTFDC1 at the CASS4 locus.

Late-onset Alzheimer's disease (LOAD) research has principally focused on neurons over the years due to their known role in the production of amyloid beta plaques and neurofibrillary tangles. In contrast, recent genomic studies of LOAD have implicated microglia as culprits of the prolonged inflammation exacerbating the neurodegeneration observed in patient brains. Indeed, recent LOAD genome-wide association studies (GWAS) have reported multiple loci near genes related to microglial function, including TREM2, ABI3, and CR1. However, GWAS alone cannot pinpoint underlying causal variants or effector genes at such loci, as most signals reside in non-coding regions of the genome and could presumably confer their influence frequently via long-range regulatory interactions. We elected to carry out a combination of ATAC-seq and high-resolution promoter-focused Capture-C in two human microglial cell models (iPSC-derived microglia and HMC3) in order to physically map interactions between LOAD GWAS-implicated candidate causal variants and their corresponding putative effector genes. Notably, we observed consistent evidence that rs6024870 at the GWAS CASS4 locus contacted the promoter of nearby gene, RTFDC1. We subsequently observed a directionallly consistent decrease in RTFDC1 expression with the the protective minor A allele of rs6024870 via both luciferase assays in HMC3 cells and expression studies in primary human microglia. Through CRISPR-Cas9-mediated deletion of the putative regulatory region harboring rs6024870 in HMC3 cells, we observed increased pro-inflammatory cytokine secretion and decreased DNA double strand break repair related, at least in part, to RTFDC1 expression levels. Our variant-to-function approach therefore reveals that the rs6024870-harboring regulatory element at the LOAD 'CASS4' GWAS locus influences both microglial inflammatory capacity and DNA damage resolution, along with cumulative evidence implicating RTFDC1 as a novel candidate effector gene.

Hypermetabolic state is associated with circadian rhythm disruption in mouse and human cancer cells.

Crosstalk between metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to disease. Here, we investigated whether maintenance of circadian rhythms depends on specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to signal from a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function across a series of pancreatic adenocarcinoma cell lines. Metabolic profiling of congenic tumor cell clones revealed substantial diversity among these lines that we used to identify clones to generate circadian reporter lines. We observed diverse circadian profiles among these lines that varied with their metabolic phenotype: The most hypometabolic line [exhibiting low levels of oxidative phosphorylation (OxPhos) and glycolysis] had the strongest rhythms, while the most hypermetabolic line had the weakest rhythms. Pharmacological enhancement of OxPhos decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, inhibition of OxPhos enhanced circadian rhythms only in the tumor cell line in which glycolysis was also low, thereby establishing a hypometabolic state. We further analyzed metabolic and circadian phenotypes across a panel of human patient-derived melanoma cell lines and observed a significant negative association between metabolic activity and circadian cycling strength. Together, these findings suggest that metabolic heterogeneity in cancer directly contributes to circadian function and that high levels of glycolysis or OxPhos independently disrupt circadian rhythms in these cells.

Variant-to-function analysis of the childhood obesity chr12q13 locus implicates rs7132908 as a causal variant within the 3' UTR of FAIM2.

The ch12q13 locus is among the most significant childhood obesity loci identified in genome-wide association studies. This locus resides in a non-coding region within FAIM2; thus, the underlying causal variant(s) presumably influence disease susceptibility via cis-regulation. We implicated rs7132908 as a putative causal variant by leveraging our in-house 3D genomic data and public domain datasets. Using a luciferase reporter assay, we observed allele-specific cis-regulatory activity of the immediate region harboring rs7132908. We generated isogenic human embryonic stem cell lines homozygous for either rs7132908 allele to assess changes in gene expression and chromatin accessibility throughout a differentiation to hypothalamic neurons, a key cell type known to regulate feeding behavior. The rs7132908 obesity risk allele influenced expression of FAIM2 and other genes and decreased the proportion of neurons produced by differentiation. We have functionally validated rs7132908 as a causal obesity variant that temporally regulates nearby effector genes and influences neurodevelopment and survival.

Hypermetabolic state is associated with circadian rhythm disruption in mouse and human cancer cells.

Crosstalk between cellular metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to degenerative disease, including cancer. Here, we investigated whether maintenance of circadian rhythms depends upon specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to overall levels of a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function in an in vitro mouse model of pancreatic adenocarcinoma. Metabolic profiling of a library of congenic tumor cell clones revealed significant differences in levels of lactate, pyruvate, ATP, and other crucial metabolites that we used to identify candidate clones with which to generate circadian reporter lines. Despite the shared genetic background of the clones, we observed diverse circadian profiles among these lines that varied with their metabolic phenotype: the most hypometabolic line had the strongest circadian rhythms while the most hypermetabolic line had the weakest rhythms. Treatment of these tumor cell lines with bezafibrate, a peroxisome proliferator-activated receptor (PPAR) agonist shown to increase OxPhos, decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, treatment with the Complex I antagonist rotenone enhanced circadian rhythms only in the tumor cell line in which glycolysis was also low, thereby establishing a hypometabolic state. We further analyzed metabolic and circadian phenotypes across a panel of human patient-derived melanoma cell lines and observed a significant negative association between metabolic activity and circadian cycling strength. Together, these findings suggest that metabolic heterogeneity in cancer directly contributes to circadian function, and that high levels of glycolysis or OxPhos independently disrupt circadian rhythms in these cells.

Variant-to-function analysis of the childhood obesity chr12q13 locus implicates rs7132908 as a causal variant within the 3' UTR of FAIM2.

The ch12q13 obesity locus is among the most significant childhood obesity loci identified in genome-wide association studies. This locus resides in a non-coding region within FAIM2; thus, the underlying causal variant(s) presumably influence disease susceptibility via an influence on cis-regulation within the genomic region. We implicated rs7132908 as a putative causal variant at this locus leveraging a combination of our inhouse 3D genomic data, public domain datasets, and several computational approaches. Using a luciferase reporter assay in human primary astrocytes, we observed allele-specific cis-regulatory activity of the immediate region harboring rs7132908. Motivated by this finding, we went on to generate isogenic human embryonic stem cell lines homozygous for either rs7132908 allele with CRISPR-Cas9 homology-directed repair to assess changes in gene expression due to genotype and chromatin accessibility throughout a differentiation to hypothalamic neurons, a key cell type known to regulate feeding behavior. We observed that the rs7132908 obesity risk allele influenced the expression of FAIM2 along with other genes, decreased the proportion of neurons produced during differentiation, up-regulated cell death gene sets, and conversely down-regulated neuron differentiation gene sets. We have therefore functionally validated rs7132908 as a causal obesity variant which temporally regulates nearby effector genes at the ch12q13 locus and influences neurodevelopment and survival.

Neuroinflammation and EIF2 Signaling Persist despite Antiretroviral Treatment in an hiPSC Tri-culture Model of HIV Infection.

HIV-associated neurocognitive disorders (HAND) affect over half of HIV-infected individuals, despite antiretroviral therapy (ART). Therapeutically targetable mechanisms underlying HAND remain elusive, partly due to a lack of a representative model. We developed a human-induced pluripotent stem cell (hiPSC)-based model, independently differentiating hiPSCs into neurons, astrocytes, and microglia, and systematically combining to generate a tri-culture with or without HIV infection and ART. Single-cell RNA sequencing analysis on tri-cultures with HIV-infected microglia revealed inflammatory signatures in the microglia and EIF2 signaling in all three cell types. Treatment with the antiretroviral compound efavirenz (EFZ) mostly resolved these signatures. However, EFZ increased RhoGDI and CD40 signaling in the HIV-infected microglia. This activation was associated with a persistent increase in transforming growth factor α production by microglia. This work establishes a tri-culture that recapitulates key features of HIV infection in the CNS and provides a new model to examine the effects of infection, its treatment, and other co-morbid conditions.

Mitochondrial deficits in human iPSC-derived neurons from patients with 22q11.2 deletion syndrome and schizophrenia.

Schizophrenia (SZ) is a highly heterogeneous disorder in both its symptoms and risk factors. One of the most prevalent genetic risk factors for SZ is the hemizygous microdeletion at chromosome 22q11.2 (22q11DS) that confers a 25-fold increased risk. Six of the genes directly disrupted in 22qDS encode for mitochondrial-localizing proteins. Here, we test the hypothesis that stem cell-derived neurons from subjects with the 22q11DS and SZ have mitochondrial deficits relative to typically developing controls. Human iPSCs from four lines of affected subjects and five lines of controls were differentiated into forebrain-like excitatory neurons. In the patient group, we find significant reductions of ATP levels that appear to be secondary to reduced activity in oxidative phosphorylation complexes I and IV. Protein products of mitochondrial-encoded genes are also reduced. As one of the genes deleted in the 22q11.2 region is MRPL40, a component of the mitochondrial ribosome, we generated a heterozygous mutation of MRPL40 in a healthy control iPSC line. Relative to its isogenic control, this line shows similar deficits in mitochondrial DNA-encoded proteins, ATP level, and complex I and IV activity. These results suggest that in the 22q11DS MRPL40 heterozygosity leads to reduced mitochondria ATP production secondary to altered mitochondrial protein levels. Such defects could have profound effects on neuronal function in vivo.