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Alzheimer's disease is the leading cause of dementia worldwide, but the cellular pathways that underlie its pathological progression across brain regions remain poorly understood 1-3 . 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., (© 2024. The Author(s).)

Cerebrovascular dysregulation is a hallmark of Alzheimer's disease (AD), but the changes that occur in specific cell types have not been fully characterized. Here, we profile single-nucleus transcriptomes in the human cerebrovasculature in six brain regions from 220 individuals with AD and 208 age-matched controls. We annotate 22,514 cerebrovascular cells, including 11 subtypes of endothelial, pericyte, smooth muscle, perivascular fibroblast and ependymal cells. We identify 2,676 differentially expressed genes in AD, including downregulation of PDGFRB in pericytes, and of ABCB1 and ATP10A in endothelial cells, and validate the downregulation of SLC6A1 and upregulation of APOD, INSR and COL4A1 in postmortem AD brain tissues. We detect vasculature, glial and neuronal coexpressed gene modules, suggesting coordinated neurovascular unit dysregulation in AD. Integration with AD genetics reveals 125 AD differentially expressed genes directly linked to AD-associated genetic variants. Lastly, we show that APOE4 genotype-associated differences are significantly enriched among AD-associated genes in capillary and venule endothelial cells, as well as subsets of pericytes and fibroblasts., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The importance of c-MYC in regulating lymphopoiesis and promoting lymphomagenesis is well-established. Far less appreciated is the vital supporting role of MYC's relative MNT. Using Rag1Cre-mediated Mnt deletion in lymphoid progenitor cells, we show here that, during normal T cell development, MNT loss enhances apoptosis, at least in part by elevating expression of the pro-apoptotic BH3-only protein BIM. Moreover, using T lymphoma-prone VavP-MYC transgenic mice, we show that Mnt deletion reduces the pool of pre-malignant MYC-driven T lymphoid cells and abrogates thymic T lymphomagenesis. In addition, we establish that Mnt deletion prevents T lymphoma development in γ-irradiated mice, most likely by enhancing apoptosis of T lymphoid cells repopulating the depleted thymus. Taken together with our recent demonstration that MNT is vital for the survival of MYC-driven pre-malignant and malignant B lymphoid cells, these results suggest that MNT represents an important new drug target for both T and B lymphoid malignancies., (© 2023. The Author(s).)

In many human cancers the control of apoptosis is dysregulated, for instance as a result of the overexpression of pro-survival BCL-2 proteins. This promotes tumorigenesis by protecting nascent neoplastic cells from stress and renders malignant cells resistant to anti-cancer agents. Therefore, several BH3 mimetic drugs targeting distinct pro-survival proteins have been developed. The BCL-2 inhibitor Venetoclax/ABT-199, has been approved for treatment of certain blood cancers and tens of thousands of patients have already been treated effectively with this drug. To advance the clinical development of MCL-1 and BCL-XL inhibitors, a more detailed understanding of their distinct and overlapping roles in the survival of malignant as well as non-transformed cells in healthy tissues is required. Here, we discuss similarities and differences in pro-survival BCL-2 protein structure, subcellular localisation and binding affinities to the pro-apoptotic BCL-2 family members. We summarise the findings from gene-targeting studies in mice to discuss the specific roles of distinct pro-survival BCL-2 family members during embryogenesis and the survival of non-transformed cells in healthy tissues in adults. Finally, we elaborate how these findings align with or differ from the observations from the clinical development and use of BH3 mimetic drugs targeting different pro-survival BCL-2 proteins., (© 2022. Crown.)

Despite the importance of the cerebrovasculature in maintaining normal brain physiology and in understanding neurodegeneration and drug delivery to the central nervous system 1 , human cerebrovascular cells remain poorly characterized owing to their sparsity and dispersion. Here we perform single-cell characterization of the human cerebrovasculature using both ex vivo fresh tissue experimental enrichment and post mortem in silico sorting of human cortical tissue samples. We capture 16,681 cerebrovascular nuclei across 11 subtypes, including endothelial cells, mural cells and three distinct subtypes of perivascular fibroblast along the vasculature. We uncover human-specific expression patterns along the arteriovenous axis and determine previously uncharacterized cell-type-specific markers. We use these human-specific signatures to study changes in 3,945 cerebrovascular cells from patients with Huntington's disease, which reveal activation of innate immune signalling in vascular and glial cell types and a concomitant reduction in the levels of proteins critical for maintenance of blood-brain barrier integrity. Finally, our study provides a comprehensive molecular atlas of the human cerebrovasculature to guide future biological and therapeutic studies., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Hairy cell leukaemia (HCL) is a rare CD20+ B cell malignancy characterised by rare "hairy" B cells and extensive bone marrow (BM) infiltration. Frontline treatment with the purine analogue cladribine (CDA) results in a highly variable response duration. We hypothesised that analysis of the BM tumour microenvironment would identify prognostic biomarkers of response to CDA. HCL BM immunology pre and post CDA treatment and healthy controls were analysed using Digital Spatial Profiling to assess the expression of 57 proteins using an immunology panel. A bioinformatics pipeline was developed to accommodate the more complex experimental design of a spatially resolved study. Treatment with CDA was associated with the reduction in expression of HCL tumour markers (CD20, CD11c) and increased expression of myeloid markers (CD14, CD68, CD66b, ARG1). Expression of HLA-DR, STING, CTLA4, VISTA, OX40L were dysregulated pre- and post-CDA. Duration of response to treatment was associated with greater reduction in tumour burden and infiltration by CD8 T cells into the BM post-CDA. This is the first study to provide a high multiplex analysis of HCL BM microenvironment demonstrating significant immune dysregulation and identify biomarkers of response to CDA. With validation in future studies, prospective application of these biomarkers could allow early identification and increased monitoring in patients at increased relapse risk post CDA., (© 2021. The Author(s).)

Elevated platelet count is associated with poor survival in certain solid cancers, including lung cancer. In addition, experimental transplantation of cancer cell lines has uncovered a role for platelets in blood-borne metastasis. These studies, however, do not account for heterogeneity between lung cancer subtypes. Subsequently, the role of platelets in the major subtypes of non-small cell lung cancer (adenocarcinoma (ADC) and squamous cell carcinoma (SqCC)) is not fully understood. We utilised an autochthonous Kras LSL-G12D/+ ;p53 flox/flox mouse model of lung ADC together with genetic models of thrombocytopenia to interrogate the role of platelets in lung cancer growth and progression. While thrombocytopenia failed to impact primary tumour growth, in experimental metastatic models however, thrombocytopenic mice displayed significantly extended survival. Utilising a novel thrombocytopenic immunocompromised mouse, the importance of platelets in metastatic dissemination was confirmed with human KRAS-mutant ADC cell lines. Finally, retrospective analysis of a NSCLC patient cohort revealed thrombocytosis was predictive of poor survival in ADC patients with metastatic disease. Interestingly, this association was not apparent in SqCC patients. Overall, these data highlight the possibility of patient stratification using thrombocytosis as a biomarker, and indicates opportunities for potential novel treatment strategies that combine anti-platelet and lung cancer therapies.

The miR17~92 cluster plays important roles in haematopoiesis. However, it is not clear at what stage of differentiation and through which targets miR17~92 exerts this function. Therefore, we generated miR17~92 fl/fl ; RosaCreERT2 mice for inducible deletion of miR17~92 in haematopoietic cells. Bone marrow reconstitution experiments revealed that miR17~92-deleted cells were not capable to contribute to mature haematopoietic lineages, which was due to defects in haematopoietic stem/progenitor cells (HSPCs). To identify the critical factor targeted by miR17~92 we performed gene expression analysis in HSPCs, demonstrating that mRNA levels of pro-apoptotic Bim inversely correlated with the expression of the miR17~92 cluster. Strikingly, loss of pro-apoptotic BIM completely prevented the loss of HSPCs caused by deletion of miR17~92. The BIM/miR17~92 interaction is conserved in human CD34 + HSPCs, as miR17~92 inhibition or blockade of its binding to the BIM 3'UTR reduced the survival and growth of these cells. Despite the prediction that miR17~92 functions by impacting a plethora of different targets, the absence of BIM alone is sufficient to prevent all defects caused by deletion of miR17~92 in haematopoietic cells.

Many acute myeloid leukaemias (AMLs) express high levels of BCL-2 and MCL-1, especially after therapy. To test the impact of these anti-apoptotic proteins on AML development and treatment, we used haemopoietic reconstitution to generate MLL-AF9 AMLs expressing BCL-2 or Mcl-1 transgenes. AMLs with elevated BCL-2 or MCL-1 had a higher proportion of mature myeloid cells but, like conventional MLL-AF9 AMLs, were readily transplantable. Short-term cell lines established from multiple primary AMLs of each genotype were tested in vitro for susceptibility to chemotherapeutics currently used for treating AML (daunorubicin, etoposide, cytarabine); the proteasome inhibitor bortezomib; CDK7/9 inhibitors; and BH3 mimetics, which bind and inhibit pro-survival proteins. The BH3 mimetics tested, alone and in combination with the other drugs, were: ABT-737 which, like its clinical counterpart navitoclax, targets BCL-2, BCL-X L and BCL-W; BCL-2-specific ABT-199 (venetoclax); BCL-X L -specific A-1331852; and S63845, a new MCL-1-specific BH3 mimetic. As single agents, daunorubicin and bortezomib had the greatest efficacy. Elevated MCL-1 or BCL-2 reduced sensitivity to daunorubicin but, surprisingly, not to bortezomib. MCL-1 markedly enhanced resistance to ABT-737 and ABT-199 but not S63845, and BCL-2 increased resistance to S63845 but not to ABT-737 or ABT-199. Notable synergies were achieved by combining BH3 mimetics with daunorubicin: S63845 increased the sensitivity of both MCL-1 and BCL-2 overexpressing MLL-AF9 AMLs, and ABT-737 aided in killing those overexpressing BCL-2. Synergy between daunorubicin and ABT-199 was also apparent in vivo, although not curative. Impressive synergistic responses were achieved for human MLL-fusion AML cell lines treated with daunorubicin plus either ABT-737, ABT-199 or S63845, and with ABT-199 plus S63845, with or without daunorubicin. Our data suggest that AML patients may benefit from combining conventional cytotoxic drugs with BH3 mimetics targeting BCL-2 or MCL-1 or, if tolerated, both these agents.

RIPK1 is an essential downstream component of many pattern recognition and death receptors. RIPK1 can promote the activation of caspase-8 induced apoptosis and RIPK3-MLKL-mediated necroptosis, however, during development RIPK1 limits both forms of cell death. Accordingly, Ripk1 -/- mice present with systemic cell death and consequent multi-organ inflammation, which is driven through the activation of both FADD-caspase-8 and RIPK3-MLKL signaling pathways causing perinatal lethality. TRADD is a death domain (DD) containing molecule that mediates signaling downstream of TNFR1 and the TLRs. Following the disassembly of the upstream receptor complexes either RIPK1 or TRADD can form a complex with FADD-caspase-8-cFLIP, via DD-DD interactions with FADD, facilitating the activation of caspase-8. We show that genetic deletion of Ripk1 licenses TRADD to complex with FADD-caspase-8 and activates caspase-8 during development. Deletion of Tradd provided no survival advantage to Ripk1 -/- animals and yet was sufficient to reduce the systemic cell death and inflammation, rescue the intestinal and thymic histopathologies, reduce cleaved caspases in most tissues and rescue the anemia observed in Ripk1 -/- neonates. Furthermore, deletion of Ripk3 is sufficient to rescue the neonatal lethality of Ripk1 -/- Tradd -/- animals and delays but does not completely prevent early mortality. Although Ripk3 deletion provides a significant survival advantage, Ripk1 -/- Tradd -/- Ripk3 -/- animals die between 22 and 49 days, are runty compared to littermate controls and present with splenomegaly. These findings reveal a new mechanism by which RIPK1 limits apoptosis through blocking TRADD recruitment to FADD and preventing aberrant activation of caspase-8.

Thrombopoietin (TPO) is the master cytokine regulator of megakaryopoiesis. In addition to regulation of megakaryocyte and platelet number, TPO is important for maintaining proper hematopoietic stem cell (HSC) function. It was previously shown that a number of lymphoid genes were upregulated in HSCs from Tpo -/- mice. We investigated if absent or enhanced TPO signaling would influence normal B-lymphopoiesis. Absent TPO signaling in Mpl -/- mice led to enrichment of a common lymphoid progenitor (CLP) signature in multipotential lineage-negative Sca-1 + c-Kit + (LSK) cells and an increase in CLP formation. Moreover, Mpl -/- mice exhibited increased numbers of PreB2 and immature B-cells in bone marrow and spleen, with an increased proportion of B-lymphoid cells in the G1 phase of the cell cycle. Conversely, elevated TPO signaling in Tpo Tg mice was associated with reduced B-lymphopoiesis. Although at steady state, peripheral blood lymphocyte counts were normal in both models, Mpl -/- Eµ-myc mice showed an enhanced preneoplastic phase with increased numbers of splenic PreB2 and immature B-cells, a reduced quiescent fraction, and augmented blood lymphocyte counts. Thus, although Mpl is not expressed on lymphoid cells, TPO signaling may indirectly influence B-lymphopoiesis and the preneoplastic state in Myc-driven B-cell lymphomagenesis by lineage priming in multipotential progenitor cells.

The discovery and characterisation of haematopoietic stem cells has required decades of research. The identification of adult bone marrow as a source of haematopoietic cells capable of protecting an organism from otherwise lethal irradiation led to the intense search for their identity and characteristics. Using functional assays along with evolving techniques for isolation of haematopoietic cells, haematopoietic stem cell populations were able to be enriched and their characteristics analysed. The key haematopoietic stem cell characteristics of pluripotentiality and the ability for self-renewal have emerged as characteristics of several haematopoietic stem cell populations, including those that have recently challenged the conventional concepts of the haematopoietic hierarchy. Human allogeneic stem cell therapy relies on these functional characteristics of haematopoietic stem cells that can be isolated from peripheral blood, bone marrow or cord blood, with the additional requirement that immunological barriers need to be overcome to allow sustained engraftment while minimising risk of graft-versus-host disease developing in the recipient of transplanted stem cells. Current and future research will continue to focus on the identification of haematopoietic stem cell regulators and methods for in vitro and in vivo stem cell manipulation, including genome editing, to expand the scope, potential and safety of therapy using haematopoietic stem cells.