Intracellular cholesterol visualization in brain tissue using D4H∗.

Studying cholesterol biology in the brain has been greatly hindered by the lack of adequate cholesterol visualization techniques. Here, we present a protocol for using a high-affinity cholesterol probe D4H∗-mCherry as a histology reagent in mouse or human brain tissue. We describe steps for D4H∗ tissue treatment and crosslinking leading to stable labeling of intracellular membrane cholesterol. Furthermore, co-labeling with Rab5 endosomal marker and optimized buffers to reduce background enable punctate cholesterol visualization within the organelle membranes.

Isoform- and cell-state-specific lipidation of ApoE in astrocytes.

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.

APOE2, E3, and E4 differentially modulate cellular homeostasis, cholesterol metabolism, and inflammatory response in isogenic iPSC-derived astrocytes.

The apolipoprotein E4 (APOE4) variant is the strongest genetic risk factor for Alzheimer disease (AD), while the APOE2 allele is protective. A major question is how different APOE genotypes affect the physiology of astrocytes, the main APOE-producing brain cells. Here, we differentiated human APOE-isogenic induced pluripotent stem cells (iPSCs) (APOE4, E3, E2, and APOE knockout [APOE-KO]) to functional "iAstrocytes". Mass-spectrometry-based proteomic analysis showed genotype-dependent reductions of cholesterol and lipid metabolic and biosynthetic pathways (reduction: APOE4 >E3 >E2). Cholesterol efflux and biosynthesis were reduced in APOE4 iAstrocytes, while subcellular localization of cholesterol in lysosomes was elevated. An increase in immunoregulatory proteomic pathways (APOE4 >E3 >E2) was accompanied by elevated cytokine release in APOE4 cells (APOE4 >E3 >E2 >KO). Activation of iAstrocytes exacerbated proteomic changes and cytokine secretion mostly in APOE4 iAstrocytes, while APOE2 and APOE-KO iAstrocytes were least affected. Taken together, APOE4 iAstrocytes reveal a disease-relevant phenotype, causing dysregulated cholesterol/lipid homeostasis, increased inflammatory signaling, and reduced ß-amyloid uptake, while APOE2 iAstrocytes show opposing effects.

Increased maturation of iPSC-derived neurons in a hydrogel-based 3D culture.

BACKGROUND: Induced pluripotent stem cells (iPSCs) can be differentiated into virtually every desired cell type, offering significant potential for modeling human diseases in vitro. A disadvantage is that iPSC-derived cells represent an immature, which presents a major limitation for modeling age-related diseases such as Alzheimer's disease. Evidence suggests that culturing iPSC neurons in a 3D environment may increase neuronal maturity. However, current 3D cell culture systems are cumbersome and time-consuming. NEW METHOD: We cultured iPSC-derived excitatory neurons in 3D precast hydrogel plates and compared their maturation to 2D monolayer cultures. COMPARISON WITH EXISTING METHODS: In contrast to other hydrogel-based 3D culture techniques, which require full encapsulation of cells, our hydrogel allows the seeded iPSCs and iPSC neurons to simply infiltrate the gel. RESULTS: IPSC-neurons grew to a depth of 500 µm into the hydrogel. Cell viability was comparable to 2D cultures over the course of three weeks, with even better neuronal survival in 3D cultures at the one-week time point. Levels of neuronal and synaptic maturation markers, namely, neural cell adhesion molecule 1 (NCAM1) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR2, were strongly increased in 3D cultures. Furthermore, we identified 4-repeat (4R) tau in 3D cultures, which was not detectable in 2D cultures. CONCLUSIONS: We describe a simple, hydrogel-based method for 3D iPSC culture that can serve as a fast and drug-screening-compatible platform to identify new mechanisms and therapeutic targets for brain diseases. We further provided evidence for the increased maturation of iPSC neurons in a 3D microenvironment.

Neurite Collapse and Altered ER Ca2+ Control in Human Parkinson Disease Patient iPSC-Derived Neurons with LRRK2 G2019S Mutation.

The Parkinson disease (PD) genetic LRRK2 gain-of-function mutations may relate to the ER pathological changes seen in PD patients at postmortem. Human induced pluripotent stem cell (iPSC)-derived neurons with the PD pathogenic LRRK2 G2019S mutation exhibited neurite collapse when challenged with the ER Ca2+ influx sarco/ER Ca2+-ATPase inhibitor thapsigargin (THP). Baseline ER Ca2+ levels measured with the ER Ca2+ indicator CEPIA-ER were lower in LRRK2 G2019S human neurons, including in differentiated midbrain dopamine neurons in vitro. After THP challenge, PD patient-derived neurons displayed increased Ca2+ influx and decreased intracellular Ca2+ buffering upon membrane depolarization. These effects were reversed following LRRK2 mutation correction by antisense oligonucleotides and gene editing. Gene expression analysis in LRRK2 G2019S neurons identified modified levels of key store-operated Ca2+ entry regulators, with no alterations in ER Ca2+ efflux. These results demonstrate PD gene mutation LRRK2 G2019S ER calcium-dependent pathogenic effects in human neurons.