Single-cell multiregion dissection of Alzheimer's disease.

Alzheimer's disease is the leading cause of dementia worldwide, but the cellular pathways that underlie its pathological progression across brain regions remain poorly understood1-3. Here we report a single-cell transcriptomic atlas of six different brain regions in the aged human brain, covering 1.3 million cells from 283 post-mortem human brain samples across 48 individuals with and without Alzheimer's disease. We identify 76 cell types, including region-specific subtypes of astrocytes and excitatory neurons and an inhibitory interneuron population unique to the thalamus and distinct from canonical inhibitory subclasses. We identify vulnerable populations of excitatory and inhibitory neurons that are depleted in specific brain regions in Alzheimer's disease, and provide evidence that the Reelin signalling pathway is involved in modulating the vulnerability of these neurons. We develop a scalable method for discovering gene modules, which we use to identify cell-type-specific and region-specific modules that are altered in Alzheimer's disease and to annotate transcriptomic differences associated with diverse pathological variables. We identify an astrocyte program that is associated with cognitive resilience to Alzheimer's disease pathology, tying choline metabolism and polyamine biosynthesis in astrocytes to preserved cognitive function late in life. Together, our study develops a regional atlas of the ageing human brain and provides insights into cellular vulnerability, response and resilience to Alzheimer's disease pathology.

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.

Transcriptomic and spatial dissection of human ex vivo right atrial tissue reveals proinflammatory microvascular changes in ischemic heart disease.

Cardiovascular disease plays a central role in the electrical and structural remodeling of the right atrium, predisposing to arrhythmias, heart failure, and sudden death. Here, we dissect with single-nuclei RNA sequencing (snRNA-seq) and spatial transcriptomics the gene expression changes in the human ex vivo right atrial tissue and pericardial fluid in ischemic heart disease, myocardial infarction, and ischemic and non-ischemic heart failure using asymptomatic patients with valvular disease who undergo preventive surgery as the control group. We reveal substantial differences in disease-associated gene expression in all cell types, collectively suggesting inflammatory microvascular dysfunction and changes in the right atrial tissue composition as the valvular and vascular diseases progress into heart failure. The data collectively suggest that investigation of human cardiovascular disease should expand to all functionally important parts of the heart, which may help us to identify mechanisms promoting more severe types of the disease.

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.

Epigenomic dissection of Alzheimer's disease pinpoints causal variants and reveals epigenome erosion.

Recent work has identified dozens of non-coding loci for Alzheimer's disease (AD) risk, but their mechanisms and AD transcriptional regulatory circuitry are poorly understood. Here, we profile epigenomic and transcriptomic landscapes of 850,000 nuclei from prefrontal cortexes of 92 individuals with and without AD to build a map of the brain regulome, including epigenomic profiles, transcriptional regulators, co-accessibility modules, and peak-to-gene links in a cell-type-specific manner. We develop methods for multimodal integration and detecting regulatory modules using peak-to-gene linking. We show AD risk loci are enriched in microglial enhancers and for specific TFs including SPI1, ELF2, and RUNX1. We detect 9,628 cell-type-specific ATAC-QTL loci, which we integrate alongside peak-to-gene links to prioritize AD variant regulatory circuits. We report differential accessibility of regulatory modules in late AD in glia and in early AD in neurons. Strikingly, late-stage AD brains show global epigenome dysregulation indicative of epigenome erosion and cell identity loss.

Neuronal DNA double-strand breaks lead to genome structural variations and 3D genome disruption in neurodegeneration.

Persistent DNA double-strand breaks (DSBs) in neurons are an early pathological hallmark of neurodegenerative diseases including Alzheimer's disease (AD), with the potential to disrupt genome integrity. We used single-nucleus RNA-seq in human postmortem prefrontal cortex samples and found that excitatory neurons in AD were enriched for somatic mosaic gene fusions. Gene fusions were particularly enriched in excitatory neurons with DNA damage repair and senescence gene signatures. In addition, somatic genome structural variations and gene fusions were enriched in neurons burdened with DSBs in the CK-p25 mouse model of neurodegeneration. Neurons enriched for DSBs also had elevated levels of cohesin along with progressive multiscale disruption of the 3D genome organization aligned with transcriptional changes in synaptic, neuronal development, and histone genes. Overall, this study demonstrates the disruption of genome stability and the 3D genome organization by DSBs in neurons as pathological steps in the progression of neurodegenerative diseases.

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.

Polycomb contraction differentially regulates terminal human hematopoietic differentiation programs.

BACKGROUND: Lifelong production of the many types of mature blood cells from less differentiated progenitors is a hierarchically ordered process that spans multiple cell divisions. The nature and timing of the molecular events required to integrate the environmental signals, transcription factor activity, epigenetic modifications, and changes in gene expression involved are thus complex and still poorly understood. To address this gap, we generated comprehensive reference epigenomes of 8 phenotypically defined subsets of normal human cord blood. RESULTS: We describe a striking contraction of H3K27me3 density in differentiated myelo-erythroid cells that resembles a punctate pattern previously ascribed to pluripotent embryonic stem cells. Phenotypically distinct progenitor cell types display a nearly identical repressive H3K27me3 signature characterized by large organized chromatin K27-modification domains that are retained by mature lymphoid cells but lost in terminally differentiated monocytes and erythroblasts. We demonstrate that inhibition of polycomb group members predicted to control large organized chromatin K27-modification domains influences lymphoid and myeloid fate decisions of primary neonatal hematopoietic progenitors in vitro. We further show that a majority of active enhancers appear in early progenitors, a subset of which are DNA hypermethylated and become hypomethylated and induced during terminal differentiation. CONCLUSION: Primitive human hematopoietic cells display a unique repressive H3K27me3 signature that is retained by mature lymphoid cells but is lost in monocytes and erythroblasts. Intervention data implicate that control of this chromatin state change is a requisite part of the process whereby normal human hematopoietic progenitor cells make lymphoid and myeloid fate decisions.

Regulatory genomic circuitry of human disease loci by integrative epigenomics.

Annotating the molecular basis of human disease remains an unsolved challenge, as 93% of disease loci are non-coding and gene-regulatory annotations are highly incomplete1-3. Here we present EpiMap, a compendium comprising 10,000 epigenomic maps across 800 samples, which we used to define chromatin states, high-resolution enhancers, enhancer modules, upstream regulators and downstream target genes. We used this resource to annotate 30,000 genetic loci that were associated with 540 traits4, predicting trait-relevant tissues, putative causal nucleotide variants in enriched tissue enhancers and candidate tissue-specific target genes for each. We partitioned multifactorial traits into tissue-specific contributing factors with distinct functional enrichments and disease comorbidity patterns, and revealed both single-factor monotropic and multifactor pleiotropic loci. Top-scoring loci frequently had multiple predicted driver variants, converging through multiple enhancers with a common target gene, multiple genes in common tissues, or multiple genes and multiple tissues, indicating extensive pleiotropy. Our results demonstrate the importance of dense, rich, high-resolution epigenomic annotations for the investigation of complex traits.

Melanoma lentigo maligno amelanótico / Amelanotic lentigo maligna melanoma

RESUMEN El melanoma lentigo maligno es un subtipo de melanoma invasor, que se localiza en áreas fotodañadas de cara y cuello, manifestándose como una mácula no muy definida, de varios colores de la gama del marrón y negro. La variante amelanótica es infrecuente en cualquiera de los subtipos cutáneos de melanoma. Presentamos el caso de una mujer de 63 años, que presentó un melanoma lentigo maligno de subtipo amelanótico y de localización infrecuente, en miembro inferior.

SUMMARY Lentigomalignamelanoma is a subtype of invasive melanoma, located in photodamaged skin, particularly of the face and neck. It appears as an ill- defined macula, of heterogeneous color, ranging from brown to black. The amelanotic variant is infrequent in any of the cutaneous melanoma subtypes. We report the case of a 63-year-old woman with amelanoticlentigomaligna melanoma, located in her lower right limb.

Improved Cerenkov counting techniques based on a free parameter model.

In the past few years, two Cerenkov methods were developed to make activity measurements of high-energy beta emitters in liquid scintillation counters with two or three photomultiplier tubes (PMTs) possible. Both methods are based on a free parameter model and make use of the Frank and Tamm theory for the emission of Cerenkov light. In this article, additional effects are discussed and further improvements are presented. The dependence of the refractive index of water on the wavelength can now be taken into account, which has also an influence on the upper limit of the wavelength region for the production of Cerenkov light. In addition, the dependence of the PMT response on the wavelength is taken into account. Finally, it is possible to take a potential asymmetry of efficiencies in a system with three PMTs into account. To this end, three free parameters are assigned to each individual PMT and then determined by means of a downhill simplex optimization algorithm. The computed counting efficiencies for a triple-to-double coincidence ratio (TDCR) system were compared with experimental data for (32)P, (89)Sr, and (90)Y.

Extension of the TDCR model to compute counting efficiencies for radionuclides with complex decay schemes.

The triple-to-double coincidence ratio (TDCR) method is frequently used to measure the activity of radionuclides decaying by pure ß emission or electron capture (EC). Some radionuclides with more complex decays have also been studied, but accurate calculations of decay branches which are accompanied by many coincident γ transitions have not yet been investigated. This paper describes recent extensions of the model to make efficiency computations for more complex decay schemes possible. In particular, the MICELLE2 program that applies a stochastic approach of the free parameter model was extended. With an improved code, efficiencies for ß(-), ß(+) and EC branches with up to seven coincident γ transitions can be calculated. Moreover, a new parametrization for the computation of electron stopping powers has been implemented to compute the ionization quenching function of 10 commercial scintillation cocktails. In order to demonstrate the capabilities of the TDCR method, the following radionuclides are discussed: (166m)Ho (complex ß(-)/γ), (59)Fe (complex ß(-)/γ), (64)Cu (ß(-), ß(+), EC and EC/γ) and (229)Th in equilibrium with its progenies (decay chain with many α, ß and complex ß(-)/γ transitions).

Improved method for the calculation of the counting efficiency of electron-capture nuclides in liquid scintillation samples.

The methods to compute the counting efficiency of electron-capture nuclides in liquid scintillation counting have been improved in several previous studies. The main improvements comprise a more realistic treatment of the ejection of photoelectrons and subsequent rearrangement processes in the atomic shell as well as a more detailed atomic rearrangement model. The latter was realized in the MICELLE code by means of a new stochastic approach. This new model was also developed to account for energy deposits within micelles. The recent improvements have now been combined in an updated version of the MICELLE code, which also makes the computation of the counting efficiency of complex decay schemes possible. In this paper, we describe and discuss recent extensions and improvements of the models and further corrections. The calculated counting efficiencies of selected radionuclides are compared with the experimental data obtained by liquid scintillation counting. For the measurements, we use standard solutions, which were calibrated by other methods.

Monte Carlo simulation of Auger-electron spectra.

A procedure to calculate the complex spectra of electron-capture nuclides which simultaneously eject several electrons and X-rays with different energies is presented. The model is applied to compute spectra of the radionuclides (125)I, (123)I and (111)In. The spectra are then compared with experimental spectra obtained by means of liquid scintillation counting. To this end, the computed spectra were transformed to allow for the nonlinear response function for a liquid scintillator, chemical quenching, as well as the Wallac-type amplifier used for the measurements. The calculated spectra are important for applications of free parameter models in liquid scintillation counting and also for studying the impact of electron-capture nuclides on DNA.

The ionization quenching function for coincident electrons.

When two electrons interact simultaneously with a liquid scintillator, they generate two distributions of solvent excited molecules. These distributions can overlap totally, partially or not overlap at all. The overlap produces an increase of the excitation density, and consequently a modification of the ionization quenching. A theoretical description that calculates the mean overlap and the ionization quench function for multiple electron coincidences is presented. We analyse the overlapping factor for electrons of similar energies. Two overlap models, two dimensional (2D) and three dimensional (3D), have been developed. Both Birks and Voltz formulae have been applied to a KLM atomic rearrangement model to compute the ionization quenching function. The counting efficiency for (125)I obtained by applying the overlapping correction is about 1% lower than that computed without this correction.

Determination of the shape factor of (90)Sr by means of the cutoff energy yield method.

Usually, Kurie plots are used to analyze beta-spectra shape-factor functions measured by means of semiconductor and magnetic spectrometers. A drawback of these techniques is the occurrence of self-absorption within the samples through which the emission spectrum is altered. In liquid-scintillation samples self-absorption does not occur, but the poor energy resolution makes the analysis of the spectra difficult. To overcome this problem, two resolution-invariant observables are used for determining the shape-factor function of (90)Sr: (1) the maximum point energy and (2) the cutoff energy yield. The measured shape-factor function of (90)Sr agrees with the one which is predicted by theory for the first-forbidden unique transition.

Study of a Monte Carlo rearrangement model for the activity determination of electron-capture nuclides by means of liquid scintillation counting.

A new Monte Carlo approach for the computation of the electron spectra of electron-capture nuclides is applied to obtain efficiencies in liquid scintillation counting for CIEMAT/NIST applications. The new method is applied to the radionuclides (109)Cd and (125)I by using a stochastic atomic rearrangement model, taking into account rearrangement processes including L-, M-, and N-subshells. The counting efficiencies were computed with the new code MICELLE which also comprises an approach for calculating the counting efficiency of a radionuclide in a gel phase sample. The calculated counting efficiencies are compared with experimental results.

Migraineurs with patent foramen ovale have larger right-to-left shunt despite similar atrial septal characteristics.

The objective of the study was to assess differences in proportion of large right-to-left shunt (RLS) and atrial septal characteristics between migraineurs and non-migraineurs referred for transcatheter closure of patent foramen ovale (PF0). This retrospective study took place in a large metropolitan medical centre. The patients were migraineurs with aura (n=52), migraineurs without aura (n=19) and non-migraineurs (n=149). RLS was evaluated before closure using bilateral power m-mode transcranial Doppler at rest and after calibrated, sustained Valsalva manoeuvre, and graded with a validated 0-5 scale. Intracardiac echocardiography was used to assess atrial septal characteristics. Migraineurs had a higher proportion of large RLS (Grade IV or V) than nonmigraineurs at rest and after calibrated Valsalva (rest, p=0.04; Valsalva, p=0.01). Atrial septal characteristics were similar between groups. Migraine is associated with larger RLS at rest and strain; however migraine status does not predict PFO characteristics.

Analysis and simulation of the relative lethality of Auger-electron-emitting radionuclides with a liquid-scintillation counter.

PURPOSE: The efficiency of strand-break induction and the counting efficiency of a liquid-scintillation counter can both be described similarly in terms of Poisson statistics. The aim of this work is to relate these two concepts, developing a simple method to simulate with a liquid-scintillation counter the relative biological effects between two different electron-emitting radionuclides. METHODS: A gel scintillator can be used to confine the decaying nuclei into nanoscale structures of liquid water (micelles). Because the fluorescing agents of the gel lay outside the micelle structure, the low-energy electrons emitted by the decaying nucleus lose part of their energy within the micelle structure before being detected, resulting in a negative increment of the counting efficiency. The difference in the counting efficiency between two gels with micelles of different characteristic sizes is applied to simulate the relative lethality of the radionuclides. RESULTS: The results are only qualitatively successful. A better accuracy cannot be achieved for commercial liquid-scintillation spectrometers, which have two photomultiplier tubes of identical gain. Also the comparison cannot be extended to low-Z Auger-electron-emitting radionuclides such as (55)Fe, since the micelle size effect is significantly increased by the interference of the L-Auger electrons. CONCLUSIONS: A liquid-scintillation counter with a gain decreased by a factor of 2.5 in one of the two photomultiplier tubes would be necessary to improve the simulation of the damaging efficiency.