An aging-sensitive compensatory secretory phospholipase that confers neuroprotection and cognitive resilience.

Cognitive reserve theory posits a role for compensatory mechanisms in the aging brain in moderating cognitive decline and risk for Alzheimer's Disease (AD). However, the identities of such mechanisms have remained elusive. A screen for hippocampal dentate granule cell (DGC) synapse loss-induced factors identified a secreted phospholipase , Pla2g2f , whose expression increases in DGCs during aging. Pla2g2f deletion in DGCs exacerbates aging-associated pathophysiological changes including synapse loss, inflammatory microglia, reactive astrogliosis, impaired neurogenesis, lipid dysregulation and hippocampal-dependent memory loss. Conversely, boosting Pla2g2f in DGCs during aging is sufficient to preserve synapses, reduce inflammatory microglia and reactive gliosis, prevent hippocampal-dependent memory impairment and modify trajectory of cognitive decline. Ex vivo, neuronal-PLA2G2F mediates intercellular signaling to decrease lipid droplet burden in microglia. Boosting Pla2g2f expression in DGCs of an aging-sensitive AD model reduces amyloid load and improves memory. Our findings implicate PLA2G2F as a compensatory neuroprotective factor that counteracts aging-associated cognitive decline. Highlights: Pla2g2f expression is increased in DGCs following synapse loss and during aging Pla2g2f levels determine aging-associated pathophysiology and cognitive resilience PLA2G2F maintains lipid homeostasis Boosting Pla2g2f expression improves memory in an aging-sensitive AD mouse model.

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.

Multisensory gamma stimulation promotes glymphatic clearance of amyloid.

The glymphatic movement of fluid through the brain removes metabolic waste1-4. Noninvasive 40 Hz stimulation promotes 40 Hz neural activity in multiple brain regions and attenuates pathology in mouse models of Alzheimer's disease5-8. Here we show that multisensory gamma stimulation promotes the influx of cerebrospinal fluid and the efflux of interstitial fluid in the cortex of the 5XFAD mouse model of Alzheimer's disease. Influx of cerebrospinal fluid was associated with increased aquaporin-4 polarization along astrocytic endfeet and dilated meningeal lymphatic vessels. Inhibiting glymphatic clearance abolished the removal of amyloid by multisensory 40 Hz stimulation. Using chemogenetic manipulation and a genetically encoded sensor for neuropeptide signalling, we found that vasoactive intestinal peptide interneurons facilitate glymphatic clearance by regulating arterial pulsatility. Our findings establish novel mechanisms that recruit the glymphatic system to remove brain amyloid.

Single-cell atlas reveals correlates of high cognitive function, dementia, and resilience to Alzheimer's disease pathology.

Alzheimer's disease (AD) is the most common cause of dementia worldwide, but the molecular and cellular mechanisms underlying cognitive impairment remain poorly understood. To address this, we generated a single-cell transcriptomic atlas of the aged human prefrontal cortex covering 2.3 million cells from postmortem human brain samples of 427 individuals with varying degrees of AD pathology and cognitive impairment. Our analyses identified AD-pathology-associated alterations shared between excitatory neuron subtypes, revealed a coordinated increase of the cohesin complex and DNA damage response factors in excitatory neurons and in oligodendrocytes, and uncovered genes and pathways associated with high cognitive function, dementia, and resilience to AD pathology. Furthermore, we identified selectively vulnerable somatostatin inhibitory neuron subtypes depleted in AD, discovered two distinct groups of inhibitory neurons that were more abundant in individuals with preserved high cognitive function late in life, and uncovered a link between inhibitory neurons and resilience to AD pathology.

Human microglial state dynamics in Alzheimer's disease progression.

Altered microglial states affect neuroinflammation, neurodegeneration, and disease but remain poorly understood. Here, we report 194,000 single-nucleus microglial transcriptomes and epigenomes across 443 human subjects and diverse Alzheimer's disease (AD) pathological phenotypes. We annotate 12 microglial transcriptional states, including AD-dysregulated homeostatic, inflammatory, and lipid-processing states. We identify 1,542 AD-differentially-expressed genes, including both microglia-state-specific and disease-stage-specific alterations. By integrating epigenomic, transcriptomic, and motif information, we infer upstream regulators of microglial cell states, gene-regulatory networks, enhancer-gene links, and transcription-factor-driven microglial state transitions. We demonstrate that ectopic expression of our predicted homeostatic-state activators induces homeostatic features in human iPSC-derived microglia-like cells, while inhibiting activators of inflammation can block inflammatory progression. Lastly, we pinpoint the expression of AD-risk genes in microglial states and differential expression of AD-risk genes and their regulators during AD progression. Overall, we provide insights underlying microglial states, including state-specific and AD-stage-specific microglial alterations at unprecedented resolution.

Neurons burdened by DNA double-strand breaks incite microglia activation through antiviral-like signaling in neurodegeneration.

DNA double-strand breaks (DSBs) are linked to neurodegeneration and senescence. However, it is not clear how DSB-bearing neurons influence neuroinflammation associated with neurodegeneration. Here, we characterize DSB-bearing neurons from the CK-p25 mouse model of neurodegeneration using single-nucleus, bulk, and spatial transcriptomic techniques. DSB-bearing neurons enter a late-stage DNA damage response marked by nuclear factor κB (NFκB)-activated senescent and antiviral immune pathways. In humans, Alzheimer's disease pathology is closely associated with immune activation in excitatory neurons. Spatial transcriptomics reveal that regions of CK-p25 brain tissue dense with DSB-bearing neurons harbor signatures of inflammatory microglia, which is ameliorated by NFκB knockdown in neurons. Inhibition of NFκB in DSB-bearing neurons also reduces microglia activation in organotypic mouse brain slice culture. In conclusion, DSBs activate immune pathways in neurons, which in turn adopt a senescence-associated secretory phenotype to elicit microglia activation. These findings highlight a previously unidentified role for neurons in the mechanism of disease-associated neuroinflammation.

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.

Isolation and Fluorescent Labeling of Extracellular Vesicles from Cultured Tumor Cells.

Extracellular vesicles (EVs), comprising exosomes, ectosomes, and apoptotic bodies, are an important component of molecular cell-to-cell communication, and are critically involved in the pathophysiology of various diseases, including tumors. In order to study the interaction of tumor cell-derived EVs with their target cells and to investigate their biological functions in comparison to other tumor cell-released factors, efficient isolation of EVs from cultured tumor cells, as well as fluorescent labeling of these EVs, is often necessary. In addition, EVs and EV-like particles are emerging as versatile vehicles for the delivery of therapeutic substances. Here, we describe an easy size exclusion chromatography-based method to isolate EVs from the mouse melanoma cell line B16F10 that yields highly enriched EV samples for subsequent applications such as molecular and functional studies. Our protocol also includes an optional labeling step with the lipophilic dye DiD, which allows tracking of EV uptake by recipient cells in vitro and in vivo.

Melanoma-derived extracellular vesicles mediate lymphatic remodelling and impair tumour immunity in draining lymph nodes.

Tumour-draining lymph nodes (LNs) undergo massive remodelling including expansion of the lymphatic sinuses, a process that has been linked to lymphatic metastasis by creation of a pre-metastatic niche. However, the signals leading to these changes have not been completely understood. Here, we found that extracellular vesicles (EVs) derived from melanoma cells are rapidly transported by lymphatic vessels to draining LNs, where they selectively interact with lymphatic endothelial cells (LECs) as well as medullary sinus macrophages. Interestingly, uptake of melanoma EVs by LN-resident LECs was partly dependent on lymphatic VCAM-1 expression, and induced transcriptional changes as well as proliferation of those cells. Furthermore, melanoma EVs shuttled tumour antigens to LN LECs for cross-presentation on MHC-I, resulting in apoptosis induction in antigen-specific CD8+ T cells. In conclusion, our data identify EV-mediated melanoma-LN LEC communication as a new pathway involved in tumour progression and tumour immune inhibition, suggesting that EV uptake or effector mechanisms in LECs might represent a new target for melanoma therapy.