APOE4 impairs myelination via cholesterol dysregulation in oligodendrocytes.

APOE4 is the strongest genetic risk factor for Alzheimer's disease1-3. However, the effects of APOE4 on the human brain are not fully understood, limiting opportunities to develop targeted therapeutics for individuals carrying APOE4 and other risk factors for Alzheimer's disease4-8. Here, to gain more comprehensive insights into the impact of APOE4 on the human brain, we performed single-cell transcriptomics profiling of post-mortem human brains from APOE4 carriers compared with non-carriers. This revealed that APOE4 is associated with widespread gene expression changes across all cell types of the human brain. Consistent with the biological function of APOE2-6, APOE4 significantly altered signalling pathways associated with cholesterol homeostasis and transport. Confirming these findings with histological and lipidomic analysis of the post-mortem human brain, induced pluripotent stem-cell-derived cells and targeted-replacement mice, we show that cholesterol is aberrantly deposited in oligodendrocytes-myelinating cells that are responsible for insulating and promoting the electrical activity of neurons. We show that altered cholesterol localization in the APOE4 brain coincides with reduced myelination. Pharmacologically facilitating cholesterol transport increases axonal myelination and improves learning and memory in APOE4 mice. We provide a single-cell atlas describing the transcriptional effects of APOE4 on the aging human brain and establish a functional link between APOE4, cholesterol, myelination and memory, offering therapeutic opportunities for Alzheimer's disease.

The interplay between microglial states and major risk factors in Alzheimer's disease through the eyes of single-cell RNA-sequencing: beyond black and white.

Microglia constitute ~10-20% of glial cells in the adult human brain. They are the resident phagocytic immune cells of the central nervous system and play an integral role as first responders during inflammation. Microglia are commonly classified as "HM" (homeostatic), "M1" (classically activated proinflammatory), or "M2" (alternatively activated). Multiple single-cell RNA-sequencing studies suggest that this discrete classification system does not accurately and fully capture the vast heterogeneity of microglial states in the brain. In fact, a recent single-cell RNA-sequencing study showed that microglia exist along a continuous spectrum of states. This spectrum spans heterogeneous populations of homeostatic and neuropathology-associated microglia in both healthy and Alzheimer's disease (AD) mouse brains. Major risk factors, such as sex, age, and genes, modulate microglial states, suggesting that shifts along the trajectory might play a causal role in AD pathogenesis. This study provides important insight into the cellular mechanisms of AD and underlines the potential of novel cell-based therapies for AD.

Single-cell atlas of ABCA7 loss-of-function reveals impaired neuronal respiration via choline-dependent lipid imbalances.

Loss-of-function (LoF) variants in the lipid transporter ABCA7 significantly increase the risk of Alzheimer's disease (odds ratio ∼2), yet the pathogenic mechanisms and the neural cell types affected by these variants remain largely unknown. Here, we performed single-nuclear RNA sequencing of 36 human post-mortem samples from the prefrontal cortex of 12 ABCA7 LoF carriers and 24 matched non-carrier control individuals. ABCA7 LoF was associated with gene expression changes in all major cell types. Excitatory neurons, which expressed the highest levels of ABCA7, showed transcriptional changes related to lipid metabolism, mitochondrial function, cell cycle-related pathways, and synaptic signaling. ABCA7 LoF-associated transcriptional changes in neurons were similarly perturbed in carriers of the common AD missense variant ABCA7 p.Ala1527Gly (n = 240 controls, 135 carriers), indicating that findings from our study may extend to large portions of the at-risk population. Consistent with ABCA7's function as a lipid exporter, lipidomic analysis of isogenic iPSC-derived neurons (iNs) revealed profound intracellular triglyceride accumulation in ABCA7 LoF, which was accompanied by a relative decrease in phosphatidylcholine abundance. Metabolomic and biochemical analyses of iNs further indicated that ABCA7 LoF was associated with disrupted mitochondrial bioenergetics that suggested impaired lipid breakdown by uncoupled respiration. Treatment of ABCA7 LoF iNs with CDP-choline (a rate-limiting precursor of phosphatidylcholine synthesis) reduced triglyceride accumulation and restored mitochondrial function, indicating that ABCA7 LoF-induced phosphatidylcholine dyshomeostasis may directly disrupt mitochondrial metabolism of lipids. Treatment with CDP-choline also rescued intracellular amyloid ß -42 levels in ABCA7 LoF iNs, further suggesting a link between ABCA7 LoF metabolic disruptions in neurons and AD pathology. This study provides a detailed transcriptomic atlas of ABCA7 LoF in the human brain and mechanistically links ABCA7 LoF-induced lipid perturbations to neuronal energy dyshomeostasis. In line with a growing body of evidence, our study highlights the central role of lipid metabolism in the etiology of Alzheimer's disease.

Infiltrating CD8+ T cells exacerbate Alzheimer's disease pathology in a 3D human neuroimmune axis model.

Brain infiltration of peripheral immune cells and their interactions with brain-resident cells may contribute to Alzheimer's disease (AD) pathology. To examine these interactions, in the present study we developed a three-dimensional human neuroimmune axis model comprising stem cell-derived neurons, astrocytes and microglia, together with peripheral immune cells. We observed an increase in the number of T cells (but not B cells) and monocytes selectively infiltrating into AD relative to control cultures. Infiltration of CD8+ T cells into AD cultures led to increased microglial activation, neuroinflammation and neurodegeneration. Using single-cell RNA-sequencing, we identified that infiltration of T cells into AD cultures led to induction of interferon-γ and neuroinflammatory pathways in glial cells. We found key roles for the C-X-C motif chemokine ligand 10 (CXCL10) and its receptor, CXCR3, in regulating T cell infiltration and neuronal damage in AD cultures. This human neuroimmune axis model is a useful tool to study the effects of peripheral immune cells in brain disease.

Lipid accumulation induced by APOE4 impairs microglial surveillance of neuronal-network activity.

Apolipoprotein E4 (APOE4) is the greatest known genetic risk factor for developing sporadic Alzheimer's disease. How the interaction of APOE4 microglia with neurons differs from microglia expressing the disease-neutral APOE3 allele remains unknown. Here, we employ CRISPR-edited induced pluripotent stem cells (iPSCs) to dissect the impact of APOE4 in neuron-microglia communication. Our results reveal that APOE4 induces a lipid-accumulated state that renders microglia weakly responsive to neuronal activity. By examining the transcriptional signatures of APOE3 versus APOE4 microglia in response to neuronal conditioned media, we established that neuronal cues differentially induce a lipogenic program in APOE4 microglia that exacerbates pro-inflammatory signals. Through decreased uptake of extracellular fatty acids and lipoproteins, we identified that APOE4 microglia disrupts the coordinated activity of neuronal ensembles. These findings suggest that abnormal neuronal network-level disturbances observed in Alzheimer's disease patients harboring APOE4 may in part be triggered by impairment in lipid homeostasis in non-neuronal cells.

A Synergistic Engineering Approach to Build Human Brain Spheroids.

Self-assembling brain spheroids derived from human stem cells closely emulate the tangled connectivity of the human brain, recapitulate aspects of organized tissue structure, and are relatively easy to manipulate compared to other existing three-dimensional (3D) cellular models. However, current platforms generate heterogeneously sized and short-lived spheroids, which do not robustly and reproducibly model human brain development and diseases. Here, we present a method to generate large-scale arrays of homogeneously sized 3D brain spheroids derived from human-induced pluripotent stem cells (hiPSCs) or immortalized neural progenitor cells to recapitulate Alzheimer's disease (AD) pathology in vitro. When embedded in extracellular matrix, these brain spheroids develop extensive outward projection of neurites and form networks, which are mediated by thick bundles of dendrites. This array facilitates cost-effective, high-throughput drug screening and mechanistic studies to better understand human brain development and neurodegenerative conditions, such as AD .

Cervicovaginal microbiota and metabolome predict preterm birth risk in an ethnically diverse cohort.

The syndrome of spontaneous preterm birth (sPTB) presents a challenge to mechanistic understanding, effective risk stratification, and clinical management. Individual associations between sPTB, self-reported ethnic ancestry, vaginal microbiota, metabolome, and innate immune response are known but not fully understood, and knowledge has yet to impact clinical practice. Here, we used multi-data type integration and composite statistical models to gain insight into sPTB risk by exploring the cervicovaginal environment of an ethnically heterogenous pregnant population (n = 346 women; n = 60 sPTB < 37 weeks' gestation, including n = 27 sPTB < 34 weeks). Analysis of cervicovaginal samples (10-15+6 weeks) identified potentially novel interactions between risk of sPTB and microbiota, metabolite, and maternal host defense molecules. Statistical modeling identified a composite of metabolites (leucine, tyrosine, aspartate, lactate, betaine, acetate, and Ca2+) associated with risk of sPTB < 37 weeks (AUC 0.752). A combination of glucose, aspartate, Ca2+, Lactobacillus crispatus, and L. acidophilus relative abundance identified risk of early sPTB < 34 weeks (AUC 0.758), improved by stratification by ethnicity (AUC 0.835). Increased relative abundance of L. acidophilus appeared protective against sPTB < 34 weeks. By using cervicovaginal fluid samples, we demonstrate the potential of multi-data type integration for developing composite models toward understanding the contribution of the vaginal environment to risk of sPTB.

Amyloid-ß42/40 ratio drives tau pathology in 3D human neural cell culture models of Alzheimer's disease.

The relationship between amyloid-ß (Aß) species and tau pathology in Alzheimer's disease (AD) is not fully understood. Here, we provide direct evidence that Aß42/40 ratio, not total Aß level, plays a critical role in inducing neurofibrillary tangles (NTFs) in human neurons. Using 3D-differentiated clonal human neural progenitor cells (hNPCs) expressing varying levels of amyloid ß precursor protein (APP) and presenilin 1 (PS1) with AD mutations, we show that pathogenic tau accumulation and aggregation are tightly correlated with Aß42/40 ratio. Roles of Aß42/40 ratio on tau pathology are also confirmed with APP transmembrane domain (TMD) mutant hNPCs, which display differential Aß42/40 ratios without mutant PS1. Moreover, naïve hNPCs co-cultured with APP TMD I45F (high Aß42/40) cells, not with I47F cells (low Aß42/40), develop robust tau pathology in a 3D non-cell autonomous cell culture system. These results emphasize the importance of reducing the Aß42/40 ratio in AD therapy.