Some osteoblasts embed within bone matrix, change shape, and become dendrite-bearing osteocytes. The circuitry that drives dendrite formation during "osteocytogenesis" is poorly understood. Here we ...show that deletion of Sp7 in osteoblasts and osteocytes causes defects in osteocyte dendrites. Profiling of Sp7 target genes and binding sites reveals unexpected repurposing of this transcription factor to drive dendrite formation. Osteocrin is a Sp7 target gene that promotes osteocyte dendrite formation and rescues defects in Sp7-deficient mice. Single-cell RNA-sequencing demonstrates defects in osteocyte maturation in the absence of Sp7. Sp7-dependent osteocyte gene networks are associated with human skeletal diseases. Moreover, humans with a SP7
mutation show defective osteocyte morphology. Sp7-dependent genes that mark osteocytes are enriched in neurons, highlighting shared features between osteocytic and neuronal connectivity. These findings reveal a role for Sp7 and its target gene Osteocrin in osteocytogenesis, revealing that pathways that control osteocyte development influence human bone diseases.
Nearly all prostate cancer deaths are from metastatic castration-resistant prostate cancer (mCRPC), but there have been few whole-genome sequencing (WGS) studies of this disease state. We performed ...linked-read WGS on 23 mCRPC biopsy specimens and analyzed cell-free DNA sequencing data from 86 patients with mCRPC. In addition to frequent rearrangements affecting known prostate cancer genes, we observed complex rearrangements of the AR locus in most cases. Unexpectedly, these rearrangements include highly recurrent tandem duplications involving an upstream enhancer of AR in 70%–87% of cases compared with <2% of primary prostate cancers. A subset of cases displayed AR or MYC enhancer duplication in the context of a genome-wide tandem duplicator phenotype associated with CDK12 inactivation. Our findings highlight the complex genomic structure of mCRPC, nominate alterations that may inform prostate cancer treatment, and suggest that additional recurrent events in the non-coding mCRPC genome remain to be discovered.
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•Linked-read genome sequencing of mCRPC resolves haplotypes and rearrangements•CDK12 inactivation is associated with a global tandem duplication phenotype•A majority of cases have duplications of an enhancer of the androgen receptor•Progression on androgen pathway inhibitors is associated with gains in AR and AR enhancer
Linked-read genome sequencing data from patients highlight that amplification of an enhancer upstream of the androgen receptor locus is a key feature of metastatic castration-resistant prostate cancer.
The loss of dopamine (DA) neurons within the substantia nigra pars compacta (SNpc) is a defining pathological hallmark of Parkinson's disease (PD). Nevertheless, the molecular features associated ...with DA neuron vulnerability have not yet been fully identified. Here, we developed a protocol to enrich and transcriptionally profile DA neurons from patients with PD and matched controls, sampling a total of 387,483 nuclei, including 22,048 DA neuron profiles. We identified ten populations and spatially localized each within the SNpc using Slide-seq. A single subtype, marked by the expression of the gene AGTR1 and spatially confined to the ventral tier of SNpc, was highly susceptible to loss in PD and showed the strongest upregulation of targets of TP53 and NR2F2, nominating molecular processes associated with degeneration. This same vulnerable population was specifically enriched for the heritable risk associated with PD, highlighting the importance of cell-intrinsic processes in determining the differential vulnerability of DA neurons to PD-associated degeneration.
Background
The accumulation of amyloid beta is a diagnostic hallmark of Alzheimer’s disease. However, the cell types that are most sensitive to its accumulation, and the transcriptional pathways that ...are induced by amyloid beta in specific cell types, remain incompletely understood.
Method
We performed single nucleus RNAseq analysis (n = 1.1 million profiles) on frontal cortical biopsies (n = 58) from individuals undergoing shunt placement for idiopathic normal pressure hydrocephalus (iNPH). Of these 58 biopsies, 21 were positive of amyloid beta, and 8 were positive for both amyloid beta and hyperphosphorylated tau. We clustered the profiles into 92 distinct clusters, and performed an integrative analysis with 30 published human and mouse datasets. We tested for proportional loss or gain of specific populations in the presence of amyloid or amyloid + tau, and identified differentially expressed genes in each population.
Results
Activated microglial and astrocytic populations were proportionally over‐represented in the pathology‐containing biopsies. Compared with postmortem datasets, the biopsy‐derived profiles showed dramatically lower activation of immediate‐early gene signatures, suggesting stronger fidelity of measured transcriptional programs to the in vivo human cell states. Comparative analysis of microglial populations with published datasets of other diseases revealed transcriptional pathways specifically induced by amyloid accumulation. Specific subsets of layer 1 interneurons were under‐represented both in amyloid‐positive biopsies, and in published datasets generated from early (Braak stage < 4) postmortem tissue.
Conclusions
The use of fresh cortical biopsies enabled a faithful measurement and analysis of glial states induced by the accumulation of amyloid beta. Specific subsets of layer 1 interneurons are particularly sensitive to depletion by amyloid beta accumulation in frontal cortex.
Cellular perturbations underlying Alzheimer’s disease (AD) are primarily studied in human postmortem samples and model organisms. Here, we generated a single-nucleus atlas from a rare cohort of ...cortical biopsies from living individuals with varying degrees of AD pathology. We next performed a systematic cross-disease and cross-species integrative analysis to identify a set of cell states that are specific to early AD pathology. These changes—which we refer to as the early cortical amyloid response—were prominent in neurons, wherein we identified a transitional hyperactive state preceding the loss of excitatory neurons, which we confirmed by acute slice physiology on independent biopsy specimens. Microglia overexpressing neuroinflammatory-related processes also expanded as AD pathology increased. Finally, both oligodendrocytes and pyramidal neurons upregulated genes associated with β-amyloid production and processing during this early hyperactive phase. Our integrative analysis provides an organizing framework for targeting circuit dysfunction, neuroinflammation, and amyloid production early in AD pathogenesis.
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•Single-nucleus profiling of human cortex biopsies uncovers amyloid-associated states•Upper-layer pyramidal neurons show hyperactivity prior to degeneration•Microglial states correlate with pathological and clinical progression•Signatures of amyloid production identified in both neurons and oligodendrocytes
Generating single-nucleus atlas from cortical biopsies of living individuals at early stage of Alzheimer’s disease, cell states of neurons, microglia, and oligodendrocytes associated with AD pathology are identified.
Abstract
Background
Emerging genetic studies of late‐onset Alzheimer’s Disease (LOAD) implicate microglia, the brain’s resident macrophages. More than half the risk genes associated with LOAD are ...expressed in microglia, yet we know little about the underlying biology or how myeloid cells contribute to LOAD pathogenesis. Single‐cell transcriptomic studies reveal diverse microglial states in patients and mouse models; however, we lack tools to track these states and understand the impact of environmental challenges or genetic susceptibility.
Method
We used human embryonic and induced pluripotent stem cell models to gain insights into microglia. These cells can be differentiated into microglia, enabling the broad characterization of cells
in vitro
and understanding their impact on the other cell types in xenograft models, allowing us to gain biological insight into disease pathogenesis.
Result
Our lab has developed a novel human stem cell‐based platform1 which induces stem cell‐derived microglia (iMGLs) to take on diverse transcriptional signatures similar to those found in the human brain in response to exposure to brain‐relevant substrates. Moreover, we conceived a lentiviral protocol that allows for efficient genetic manipulation of microglia (>90% transduction). Building on these systems, we generated reporters of microglia state to track the expression of key microglial genes and adapted iPSC “villages”2, or pooled cultures, that combine iPSC cell lines in the same dish. Using these tools
in vitro
and in xenograft models3,4, we identified genetic regulators and functional changes of microglia states in LOAD.
Conclusion
Together, these tools represent a broad toolset that will allow us to answer some of the most pressing questions, such as when and how in disease these states are formed, what is their impact on disease progression, and how plastic they are. These tools will be invaluable to understanding the role of microglia in AD and the brain in general.
1. Dolan, M.‐J.
et al. Biorxiv
2022.05.02.490100 (2022)
2. Wells, M. F. et al.
Biorxiv
2021.11.08.467815 (2021)
3. Hasselmann, J. et al.
Neuron
103
, 1016‐1033.e10 (2019)
4. Mancuso, R. et al.
Nature Neuroscience
22
, 2111‐2116 (2019)
Microglia, the macrophages of the brain parenchyma, are key players in neurodegenerative diseases such as Alzheimer's disease. These cells adopt distinct transcriptional subtypes known as states. ...Understanding state function, especially in human microglia, has been elusive owing to a lack of tools to model and manipulate these cells. Here, we developed a platform for modeling human microglia transcriptional states in vitro. We found that exposure of human stem-cell-differentiated microglia to synaptosomes, myelin debris, apoptotic neurons or synthetic amyloid-beta fibrils generated transcriptional diversity that mapped to gene signatures identified in human brain microglia, including disease-associated microglia, a state enriched in neurodegenerative diseases. Using a new lentiviral approach, we demonstrated that the transcription factor MITF drives a disease-associated transcriptional signature and a highly phagocytic state. Together, these tools enable the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.
Neurodegenerative diseases are pathologically characterized by the induction of gliotic states and the loss of specific neurons in the human brain. A precise ascertainment of cellular states altered ...and those types of neurons that are lost has not been made in Alzheimer’s and Parkinson’s disease, the two most common forms of brain degeneration. We employed new single-cell genomic technologies to better understand the cellular alterations that occur in both these diseases. In Parkinson’s disease, we developed a method to enrich midbrain dopaminergic neuronal nuclei from postmortem human samples. Using this strategy, we profiled thousands of these neurons to identify the diversity of these different types in the midbrain. Strikingly, we found one population, confined to the ventral tier of the pars compacta, was uniquely susceptible to Parkinson’s-associated cell loss. This same population was enriched for common variant heritable risk of the disease, suggesting cell-autonomous mechanisms underlie the genetics of PD. To better understand the cellular alterations in Alzheimer’s disease before death, we profiled individuals with suspected idiopathic normal pressure hydrocephalus (iNPH) with comorbid AD pathology. We identified two microglia populations, one increased and one decreased in abundance respectively, and confirmed these alterations in postmortem AD datasets. We further identified a single interneuron subpopulation residing in the uppermost layer of the neocortex as lost especially in the earliest stages of AD pathology. Finally, we find oligodendrocytes are an unrecognized contributor to beta-amyloid pathology. Measurement of beta-amyloid from stem cell-derived human oligodendrocytes confirmed that these cells are a significant producer of this important peptide. This body of work offers compelling evidence towards identifying the specific cellular state changes in association with two common brain diseases, offering new and testable hypotheses for future biological investigations into their pathogenesis.
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