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.

Nanoparticle-Mediated Delivery of Anti-PU.1 siRNA via Localized Intracisternal Administration Reduces Neuroinflammation.

Neuroinflammation is a hallmark of neurodegenerative disorders including Alzheimer's disease (AD). Microglia, the brain's immune cells, express many of the AD-risk loci identified in genome wide association studies and present a promising target for anti-inflammatory RNA therapeutics but are difficult to transfect with current methods. Here, several lipid nanoparticle (LNP) formulations are examined, and a lead candidate that supports efficient RNA delivery in cultures of human stem cell-derived microglia-like cells (iMGLs) and animal models of neuroinflammation is identified. The lead microglia LNP (MG-LNP) formulation shows minimal toxicity and improves delivery efficiency to inflammatory iMGLs, suggesting a preference for delivery into activated microglia. Intraperitoneal injection of the MG-LNP formulation generates widespread expression of the delivered reporter construct in all organs, whereas local intracisternal injection directly into the cerebrospinal fluid leads to preferential expression in the brain. It is shown that LNP-mediated delivery of siRNA targeting the PU.1 transcription factor, a known AD-risk locus, successfully reduces PU.1 levels in iMGLs and reduces neuroinflammation in mice injected with LPS and in CK-p25 mice that mimic the chronic neuroinflammation seen in AD patients. The LNP formulation represents an effective RNA delivery vehicle when applied intrathecally and can be broadly utilized to test potential neuroinflammation-directed gene therapies.

APOE4 disrupts intracellular lipid homeostasis in human iPSC-derived glia.

The E4 allele of the apolipoprotein E gene (APOE) has been established as a genetic risk factor for many diseases including cardiovascular diseases and Alzheimer's disease (AD), yet its mechanism of action remains poorly understood. APOE is a lipid transport protein, and the dysregulation of lipids has recently emerged as a key feature of several neurodegenerative diseases including AD. However, it is unclear how APOE4 perturbs the intracellular lipid state. Here, we report that APOE4, but not APOE3, disrupted the cellular lipidomes of human induced pluripotent stem cell (iPSC)-derived astrocytes generated from fibroblasts of APOE4 or APOE3 carriers, and of yeast expressing human APOE isoforms. We combined lipidomics and unbiased genome-wide screens in yeast with functional and genetic characterization to demonstrate that human APOE4 induced altered lipid homeostasis. These changes resulted in increased unsaturation of fatty acids and accumulation of intracellular lipid droplets both in yeast and in APOE4-expressing human iPSC-derived astrocytes. We then identified genetic and chemical modulators of this lipid disruption. We showed that supplementation of the culture medium with choline (a soluble phospholipid precursor) restored the cellular lipidome to its basal state in APOE4-expressing human iPSC-derived astrocytes and in yeast expressing human APOE4 Our study illuminates key molecular disruptions in lipid metabolism that may contribute to the disease risk linked to the APOE4 genotype. Our study suggests that manipulating lipid metabolism could be a therapeutic approach to help alleviate the consequences of carrying the APOE4 allele.

Reconstruction of the human blood-brain barrier in vitro reveals a pathogenic mechanism of APOE4 in pericytes.

In Alzheimer's disease, amyloid deposits along the brain vasculature lead to a condition known as cerebral amyloid angiopathy (CAA), which impairs blood-brain barrier (BBB) function and accelerates cognitive degeneration. Apolipoprotein (APOE4) is the strongest risk factor for CAA, yet the mechanisms underlying this genetic susceptibility are unknown. Here we developed an induced pluripotent stem cell-based three-dimensional model that recapitulates anatomical and physiological properties of the human BBB in vitro. Similarly to CAA, our in vitro BBB displayed significantly more amyloid accumulation in APOE4 compared to APOE3. Combinatorial experiments revealed that dysregulation of calcineurin-nuclear factor of activated T cells (NFAT) signaling and APOE in pericyte-like mural cells induces APOE4-associated CAA pathology. In the human brain, APOE and NFAT are selectively dysregulated in pericytes of APOE4 carriers, and inhibition of calcineurin-NFAT signaling reduces APOE4-associated CAA pathology in vitro and in vivo. Our study reveals the role of pericytes in APOE4-mediated CAA and highlights calcineurin-NFAT signaling as a therapeutic target in CAA and Alzheimer's disease.

Diverse structures, functions and uses of FK506 binding proteins.

FK506 (Tacrolimus), isolated from Streptomyces tsukubaenis is a powerful immunosuppressant shown to inhibit T cell activation. FK506 mediated immunosuppression requires the formation of a complex between FK506, a FK506 binding protein (FKBP) and calcineurin. Numerous FKBPs have been identified in a wide range of species, from single celled organisms to humans. FKBPs show peptidylprolyl cis/trans isomerase (PPIase) activity and have been shown to affect a wide range of cellular processes including protein folding, receptor signaling and apoptosis. FKBPs also affect numerous biological functions in addition to immunosuppression including regulation of cardiac function, neuronal function and development and have been implicated in several diseases including cardiac disease, cancer and neurodegenerative diseases such as Alzheimer's disease. More recently, FKBPs have proven useful as molecular tools for studying protein interactions, localization and functions. This review provides an overview of the current state of knowledge of FKBPs and their numerous biological functions and uses.

Functional analysis of the NHR2 domain indicates that oligomerization of Neuralized regulates ubiquitination and endocytosis of Delta during Notch signaling.

The Notch pathway plays an integral role in development by regulating cell fate in a wide variety of multicellular organisms. A critical step in the activation of Notch signaling is the endocytosis of the Notch ligands Delta and Serrate. Ligand endocytosis is regulated by one of two E3 ubiquitin ligases, Neuralized (Neur) or Mind bomb. Neur is comprised of a C-terminal RING domain, which is required for Delta ubiquitination, and two Neur homology repeat (NHR) domains. We have previously shown that the NHR1 domain is required for Delta trafficking. Here we show that the NHR1 domain also affects the binding and internalization of Serrate. Furthermore, we show that the NHR2 domain is required for Neur function and that a point mutation in the NHR2 domain (Gly430) abolishes Neur ubiquitination activity and affects ligand internalization. Finally, we provide evidence that Neur can form oligomers in both cultured cells and fly tissues, which regulate Neur activity and, by extension, ligand internalization.

Drosophila as a model to study age-related neurodegenerative disorders: Alzheimer's disease.

Alzheimer's Disease (AD) is the most common cause of dementia in the aging population. Although a variety of drug treatments can delay the onset of disease or temporarily reduce its severity, there is currently no cure or effective long-term treatment. This therapeutic void in part reflects an incomplete understanding of the biochemical pathogenesis of this disease. Model organisms, including invertebrates, have been extensively utilized to gain insight into the molecular and cellular mechanisms underlying disease. Here, we will describe how Drosophila has been used to study the function of genes associated with AD and to develop models of this devastating disease.