Models of neurodegeneration using computational approaches

Alzheimer's disease (AD), as one of the most common neurodegenerative diseases, is characterized by the loss of neuronal dysfunction and death resulting in progressive cognitive impairment. The main histopathological hallmark of AD is the accumulation and deposition of misfolded Aβ peptide as amyloid plaques, however the precise role of Aβ toxicity in the disease pathogenesis is still unclear. Moreover, at early stages of the disease the important clinical features of the disorder, in addition to memory loss, are the disruptions of circadian rhythms and spatial disorientation. In the present work I first studied the role of Aβ toxicity by comparing the findings of genome-wide association studies in sporadic AD with the results of an RNAi screen in a transgenic C. elegans model of Aβ toxicity. The initial finding was that none of the human orthologues of these worm genes are associated with risk for sporadic Alzheimer’s disease, indicating that Aβ toxicity in the worm model may not be equivalent to sporadic AD. Nevertheless, comparing the first degree physical interactors (+1 interactome) of the GWAS and worm screen-derived gene products have uncovered 4 worm genes that have a +1 interactome overlap with the GWAS genes that is larger than one would expect by chance. Three of these genes form a chaperonin complex and the fourth gene codes for actin, a major substrate of the same chaperonin. Next I have evaluated the circadian disruptions in AD by developing a new system to simultaneously monitor the oscillations of the peripheral molecular clock and behavioural rhythms in single Drosophila. Experiments were undertaken on wild- type and Aβ-expressing flies. The results indicate the robustness of the peripheral clock is not correlated with the robustness of the circadian sleep and locomotor behaviours, indicating that the molecular clock does not directly drive behaviour. This is despite period length correlations that indicate that the underlying molecular mechanisms that generate both molecular and behavioural rhythms are the same. Rhythmicity in Aβ-expressing flies is worse than in controls. I further investigated the mechanism of spatial orientation in Drosophila. It was established that in the absence of visual stimuli the flies use self-motion cues to orientate themselves within the tubes and that in a Drosophila model of Aβ toxicity this control function is disrupted

The TRiC/CCT chaperone is implicated in Alzheimer's disease based on patient GWAS and an RNAi screen in Aβ-expressing Caenorhabditis elegans.

The human Aβ peptide causes progressive paralysis when expressed in the muscles of the nematode worm, C. elegans. We have exploited this model of Aβ toxicity by carrying out an RNAi screen to identify genes whose reduced expression modifies the severity of this locomotor phenotype. Our initial finding was that none of the human orthologues of these worm genes is identical with the genome-wide significant GWAS genes reported to date (the "white zone"); moreover there was no identity between worm screen hits and the longer list of GWAS genes which included those with borderline levels of significance (the "grey zone"). This indicates that Aβ toxicity should not be considered as equivalent to sporadic AD. To increase the sensitivity of our analysis, we then considered the physical interactors (+1 interactome) of the products of the genes in both the worm and the white+grey zone lists. When we consider these worm and GWAS gene lists we find that 4 of the 60 worm genes have a +1 interactome overlap that is larger than expected by chance. Two of these genes form a chaperonin complex, the third is closely associated with this complex and the fourth gene codes for actin, the major substrate of the same chaperonin

An integrated single-cell analysis of human adrenal cortex development

The adrenal glands synthesize and release essential steroid hormones such as cortisol and aldosterone, but many aspects of human adrenal gland development are not well understood. Here, we combined single-cell and bulk RNA sequencing, spatial transcriptomics, IHC, and micro-focus computed tomography to investigate key aspects of adrenal development in the first 20 weeks of gestation. We demonstrate rapid adrenal growth and vascularization, with more cell division in the outer definitive zone (DZ). Steroidogenic pathways favored androgen synthesis in the central fetal zone, but DZ capacity to synthesize cortisol and aldosterone developed with time. Core transcriptional regulators were identified, with localized expression of HOPX (also known as Hop homeobox/homeobox-only protein) in the DZ. Potential ligand-receptor interactions between mesenchyme and adrenal cortex were seen (e.g., RSPO3/LGR4). Growth-promoting imprinted genes were enriched in the developing cortex (e.g., IGF2, PEG3). These findings reveal aspects of human adrenal development and have clinical implications for understanding primary adrenal insufficiency and related postnatal adrenal disorders, such as adrenal tumor development, steroid disorders, and neonatal stress

Transcriptional signatures associated with persisting CD19 CAR-T cells in children with leukemia

In the context of relapsed and refractory childhood pre-B cell acute lymphoblastic leukemia (R/R B-ALL), CD19-targeting chimeric antigen receptor (CAR)-T cells often induce durable remissions, which requires the persistence of CAR-T cells. In this study, we systematically analyzed CD19 CAR-T cells of 10 children with R/R B-ALL enrolled in the CARPALL trial via high-throughput single-cell gene expression and T cell receptor sequencing of infusion products and serial blood and bone marrow samples up to 5 years after infusion. We show that long-lived CAR-T cells developed a CD4/CD8 double-negative phenotype with an exhausted-like memory state and distinct transcriptional signature. This persistence signature was dominant among circulating CAR-T cells in all children with a long-lived treatment response for which sequencing data were sufficient (4/4, 100%). The signature was also present across T cell subsets and clonotypes, indicating that persisting CAR-T cells converge transcriptionally. This persistence signature was also detected in two adult patients with chronic lymphocytic leukemia with decade-long remissions who received a different CD19 CAR-T cell product. Examination of single T cell transcriptomes from a wide range of healthy and diseased tissues across children and adults indicated that the persistence signature may be specific to long-lived CAR-T cells. These findings raise the possibility that a universal transcriptional signature of clinically effective, persistent CD19 CAR-T cells exists

Somatic mutations and single-cell transcriptomes reveal the root of malignant rhabdoid tumours.

Malignant rhabdoid tumour (MRT) is an often lethal childhood cancer that, like many paediatric tumours, is thought to arise from aberrant fetal development. The embryonic root and differentiation pathways underpinning MRT are not firmly established. Here, we study the origin of MRT by combining phylogenetic analyses and single-cell mRNA studies in patient-derived organoids. Comparison of somatic mutations shared between cancer and surrounding normal tissues places MRT in a lineage with neural crest-derived Schwann cells. Single-cell mRNA readouts of MRT differentiation, which we examine by reverting the genetic driver mutation underpinning MRT, SMARCB1 loss, suggest that cells are blocked en route to differentiating into mesenchyme. Quantitative transcriptional predictions indicate that combined HDAC and mTOR inhibition mimic MRT differentiation, which we confirm experimentally. Our study defines the developmental block of MRT and reveals potential differentiation therapies

Babesia duncani multi-omics identifies virulence factors and drug targets

Babesiosis is a malaria-like disease in humans and animals that is caused by Babesia species, which are tick-transmitted apicomplexan pathogens. Babesia duncani causes severe to lethal infection in humans, but despite the risk that this parasite poses as an emerging pathogen, little is known about its biology, metabolic requirements or pathogenesis. Unlike other apicomplexan parasites that infect red blood cells, B. duncani can be continuously cultured in vitro in human erythrocytes and can infect mice resulting in fulminant babesiosis and death. We report comprehensive, detailed molecular, genomic, transcriptomic and epigenetic analyses to gain insights into the biology of B. duncani. We completed the assembly, 3D structure and annotation of its nuclear genome, and analysed its transcriptomic and epigenetics profiles during its asexual life cycle stages in human erythrocytes. We used RNA-seq data to produce an atlas of parasite metabolism during its intraerythrocytic life cycle. Characterization of the B. duncani genome, epigenome and transcriptome identified classes of candidate virulence factors, antigens for diagnosis of active infection and several attractive drug targets. Furthermore, metabolic reconstitutions from genome annotation and in vitro efficacy studies identified antifolates, pyrimethamine and WR-99210 as potent inhibitors of B. duncani to establish a pipeline of small molecules that could be developed as effective therapies for the treatment of human babesiosis.We thank R. Gao for her contribution to the initial eforts to sequence the B. duncani genome. C.B.M.’s research was supported by grants from the National Institutes of Health (AI097218, GM110506, AI123321 and R43AI136118), the Steven and Alexandra Cohen Foundation (Lyme 62 2020), and the Global Lyme Alliance. S.L.’s research was supported by grants by the US National Science Foundation (IIS 1814359) and the National Institutes of Health (1R01AI169543-01). K.G.L.R.’s research was supported by the National Institutes of Allergy and Infectious Diseases (R01 AI136511, R01 AI142743-01 and R21 AI142506-01), the University of California, Riverside (NIFA-Hatch-225935) and the Health Institute Carlos III (PI20CIII/00037).S

Single cell derived mRNA signals across human kidney tumors.

Funder: Department of HealthTumor cells may share some patterns of gene expression with their cell of origin, providing clues into the differentiation state and origin of cancer. Here, we study the differentiation state and cellular origin of 1300 childhood and adult kidney tumors. Using single cell mRNA reference maps of normal tissues, we quantify reference "cellular signals" in each tumor. Quantifying global differentiation, we find that childhood tumors exhibit fetal cellular signals, replacing the presumption of "fetalness" with a quantitative measure of immaturity. By contrast, in adult cancers our assessment refutes the suggestion of dedifferentiation towards a fetal state in most cases. We find an intimate connection between developmental mesenchymal populations and childhood renal tumors. We demonstrate the diagnostic potential of our approach with a case study of a cryptic renal tumor. Our findings provide a cellular definition of human renal tumors through an approach that is broadly applicable to human cancer

Precise identification of cancer cells from allelic imbalances in single cell transcriptomes.

A fundamental step of tumour single cell mRNA analysis is separating cancer and non-cancer cells. We show that the common approach to separation, using shifts in average expression, can lead to erroneous biological conclusions. By contrast, allelic imbalances representing copy number changes directly detect the cancer genotype and accurately separate cancer from non-cancer cells. Our findings provide a definitive approach to identifying cancer cells from single cell mRNA sequencing data

Worm genes +1 interactome.

<p>The +1 interaction network for the human orthologues of the 60 worm genes that were highlighted in our worm RNAi screen. For the 60 worm-screen genes with human orthologues, there were 3191 interactions in the +1 interactome.</p

Somatic mutations and single-cell transcriptomes reveal the root of malignant rhabdoid tumours.

Malignant rhabdoid tumour (MRT) is an often lethal childhood cancer that, like many paediatric tumours, is thought to arise from aberrant fetal development. The embryonic root and differentiation pathways underpinning MRT are not firmly established. Here, we study the origin of MRT by combining phylogenetic analyses and single-cell mRNA studies in patient-derived organoids. Comparison of somatic mutations shared between cancer and surrounding normal tissues places MRT in a lineage with neural crest-derived Schwann cells. Single-cell mRNA readouts of MRT differentiation, which we examine by reverting the genetic driver mutation underpinning MRT, SMARCB1 loss, suggest that cells are blocked en route to differentiating into mesenchyme. Quantitative transcriptional predictions indicate that combined HDAC and mTOR inhibition mimic MRT differentiation, which we confirm experimentally. Our study defines the developmental block of MRT and reveals potential differentiation therapies