Deficiency in origin licensing proteins impairs cilia formation: implications for the aetiology of meier-gorlin syndrome

Mutations in ORC1, ORC4, ORC6, CDT1, and CDC6, which encode proteins required for DNA replication origin licensing, cause Meier-Gorlin syndrome (MGS), a disorder conferring microcephaly, primordial dwarfism, underdeveloped ears, and skeletal abnormalities. Mutations in ATR, which also functions during replication, can cause Seckel syndrome, a clinically related disorder. These findings suggest that impaired DNA replication could underlie the developmental defects characteristic of these disorders. Here, we show that although origin licensing capacity is impaired in all patient cells with mutations in origin licensing component proteins, this does not correlate with the rate of progression through S phase. Thus, the replicative capacity in MGS patient cells does not correlate with clinical manifestation. However, ORC1-deficient cells from MGS patients and siRNA-mediated depletion of origin licensing proteins also have impaired centrosome and centriole copy number. As a novel and unexpected finding, we show that they also display a striking defect in the rate of formation of primary cilia. We demonstrate that this impacts sonic hedgehog signalling in ORC1-deficient primary fibroblasts. Additionally, reduced growth factor-dependent signaling via primary cilia affects the kinetics of cell cycle progression following cell cycle exit and re-entry, highlighting an unexpected mechanism whereby origin licensing components can influence cell cycle progression. Finally, using a cell-based model, we show that defects in cilia function impair chondroinduction. Our findings raise the possibility that a reduced efficiency in forming cilia could contribute to the clinical features of MGS, particularly the bone development abnormalities, and could provide a new dimension for considering developmental impacts of licensing deficiency

Absence of microglia promotes diverse pathologies and early lethality in Alzheimer’s disease mice

Microglia are strongly implicated in the development and progression of Alzheimer's disease (AD), yet their impact on pathology and lifespan remains unclear. Here we utilize a CSF1R hypomorphic mouse to generate a model of AD that genetically lacks microglia. The resulting microglial-deficient mice exhibit a profound shift from parenchymal amyloid plaques to cerebral amyloid angiopathy (CAA), which is accompanied by numerous transcriptional changes, greatly increased brain calcification and hemorrhages, and premature lethality. Remarkably, a single injection of wild-type microglia into adult mice repopulates the microglial niche and prevents each of these pathological changes. Taken together, these results indicate the protective functions of microglia in reducing CAA, blood-brain barrier dysfunction, and brain calcification. To further understand the clinical implications of these findings, human AD tissue and iPSC-microglia were examined, providing evidence that microglia phagocytose calcium crystals, and this process is impaired by loss of the AD risk gene, TREM2

Uncovering the molecular architecture of Alzheimer’s disease

Alzheimer’s disease (AD) is a devastating, progressive neurodegenerative disorder that results in dementia, with care for those with dementia estimated to cost the U.S. $321 billion in 2022. Although many years of research have uncovered notable findings about AD biology, it is clear that we still have a limited understanding of the disease as evidenced by the lack of effective therapeutics against AD. For example, genome-wide association studies (GWAS) have uncovered multiple genetic risk variants, revealing novel genes and pathways for study in AD. However, it remains a challenge to ascertain the functional significance of these risk variants. Multiple studies have attempted to clarify the role of AD risk variants with “bulk”-tissue RNA- sequencing (RNA-seq), but they have been hindered by the vast cellular heterogeneity of the brain. Single-cell (scRNA-seq) and single-nucleus RNA-seq (snRNA-seq) performed on the 5XFAD mouse model of AD, as well as human AD samples, identified disease-associated glial subpopulations, but it is unclear what regulates these subtypes. Additionally, studies have revealed region-specific glial subpopulations existing in the healthy brain, suggesting potential regional differences in the disease phenotype. This dissertation aimed to clarify the molecular landscape of AD with spatial and cellular resolution with an emphasis on the analysis and integration of different data modalities and model systems.We first sought to define the cell-type specific gene regulatory programs dysregulated in AD to identify potential regulators of disease-associated cell subpopulations and to further unravel AD genetic risk (Chapter Two). Recent advances in sequencing methods now allow interrogation of the transcriptome and epigenome at the single cell resolution, and AD epigenetic data has been limited. We generated paired single-nucleus transcriptomic and epigenomic data from postmortem human brain tissue of late-stage AD and cognitively healthy controls. In addition to being the first epigenetic dataset of human AD with single-cell resolution, we directly integrated the two different data modalities, allowing us to define disease-associated glial subpopulations at the transcriptome and epigenome. We identified cell-type specific, disease-associated candidate cis-regulatory elements (cCREs) and their candidate target genes. Although this is possible with single-cell epigenetic data alone, paired gene expression data provides additional functional evidence of cCREs. We also revealed cell-type specific transcription factors dysregulated with disease, like SREBF1 in oligodendrocytes, altogether identifying both cis- and trans-gene regulatory mechanisms that may regulate AD cell states. Furthermore, we characterized the cis- regulatory landscape at AD GWAS loci in specific cell-types, providing insight into the cell-types relevant to specific AD risk variants. On the other hand, while recent transcriptomic studies have revealed both brain region- and cell- type-specific gene expression changes in AD, “bulk”-tissue and scRNA-seq do not retain spatial information for gene expression changes without careful microdissection and sequencing separate samples. The human brain’s spatially complexity at both macro- and microscopic levels underlies brain function and thus is a critical feature to consider in disease pathophysiology. We generated spatial transcriptomic data from postmortem human brain tissue from cognitively healthy controls, early-, and late-stage AD to investigate the spatial relationship of disease-associated transcriptomic changes (Chapter Three). Additionally, we performed a comparative analysis of AD in Down Syndrome (DS) and the general population by generating both spatial and single-nucleus transcriptomic data from AD in DS. To date, no published spatial or single- nucleus studies have explored the concordance between these two populations, although AD in DS may serve as an advantageous group for preclinical and clinical studies of AD. We identified regional and cell-type specific transcriptomic changes shared between both AD populations. Further, we present a time-course analysis of the spatial transcriptome of the amyloid mouse model 5XFAD to assess the AD transcriptome across multiple brain regions and identify evolutionary-conserved gene expression changes. In addition to surveying different model systems, we integrated imaging data with spatial transcriptomic data. Amyloid beta (Aβ) pathology is one of the classical hallmarks of AD and proposed as a critical driver of AD pathogenesis (amyloid cascade hypothesis). We identified transcripts spatially localized to Aβ pathology conserved between both human and mouse. We also integrated spatial and single-nucleus transcriptomic data to discover spatially defined cell signaling pathways dysregulated with disease and highlight regional heterogeneity of astrocytes. Altogether, this dissertation reveals regional and cellular molecular changes occurring in AD and contextualizes them in a systems-level framework to uncover pathways for further study in AD

Uncovering the molecular architecture of Alzheimer’s disease

Alzheimer’s disease (AD) is a devastating, progressive neurodegenerative disorder that results in dementia, with care for those with dementia estimated to cost the U.S. $321 billion in 2022. Although many years of research have uncovered notable findings about AD biology, it is clear that we still have a limited understanding of the disease as evidenced by the lack of effective therapeutics against AD. For example, genome-wide association studies (GWAS) have uncovered multiple genetic risk variants, revealing novel genes and pathways for study in AD. However, it remains a challenge to ascertain the functional significance of these risk variants. Multiple studies have attempted to clarify the role of AD risk variants with “bulk”-tissue RNA- sequencing (RNA-seq), but they have been hindered by the vast cellular heterogeneity of the brain. Single-cell (scRNA-seq) and single-nucleus RNA-seq (snRNA-seq) performed on the 5XFAD mouse model of AD, as well as human AD samples, identified disease-associated glial subpopulations, but it is unclear what regulates these subtypes. Additionally, studies have revealed region-specific glial subpopulations existing in the healthy brain, suggesting potential regional differences in the disease phenotype. This dissertation aimed to clarify the molecular landscape of AD with spatial and cellular resolution with an emphasis on the analysis and integration of different data modalities and model systems.We first sought to define the cell-type specific gene regulatory programs dysregulated in AD to identify potential regulators of disease-associated cell subpopulations and to further unravel AD genetic risk (Chapter Two). Recent advances in sequencing methods now allow interrogation of the transcriptome and epigenome at the single cell resolution, and AD epigenetic data has been limited. We generated paired single-nucleus transcriptomic and epigenomic data from postmortem human brain tissue of late-stage AD and cognitively healthy controls. In addition to being the first epigenetic dataset of human AD with single-cell resolution, we directly integrated the two different data modalities, allowing us to define disease-associated glial subpopulations at the transcriptome and epigenome. We identified cell-type specific, disease-associated candidate cis-regulatory elements (cCREs) and their candidate target genes. Although this is possible with single-cell epigenetic data alone, paired gene expression data provides additional functional evidence of cCREs. We also revealed cell-type specific transcription factors dysregulated with disease, like SREBF1 in oligodendrocytes, altogether identifying both cis- and trans-gene regulatory mechanisms that may regulate AD cell states. Furthermore, we characterized the cis- regulatory landscape at AD GWAS loci in specific cell-types, providing insight into the cell-types relevant to specific AD risk variants. On the other hand, while recent transcriptomic studies have revealed both brain region- and cell- type-specific gene expression changes in AD, “bulk”-tissue and scRNA-seq do not retain spatial information for gene expression changes without careful microdissection and sequencing separate samples. The human brain’s spatially complexity at both macro- and microscopic levels underlies brain function and thus is a critical feature to consider in disease pathophysiology. We generated spatial transcriptomic data from postmortem human brain tissue from cognitively healthy controls, early-, and late-stage AD to investigate the spatial relationship of disease-associated transcriptomic changes (Chapter Three). Additionally, we performed a comparative analysis of AD in Down Syndrome (DS) and the general population by generating both spatial and single-nucleus transcriptomic data from AD in DS. To date, no published spatial or single- nucleus studies have explored the concordance between these two populations, although AD in DS may serve as an advantageous group for preclinical and clinical studies of AD. We identified regional and cell-type specific transcriptomic changes shared between both AD populations. Further, we present a time-course analysis of the spatial transcriptome of the amyloid mouse model 5XFAD to assess the AD transcriptome across multiple brain regions and identify evolutionary-conserved gene expression changes. In addition to surveying different model systems, we integrated imaging data with spatial transcriptomic data. Amyloid beta (Aβ) pathology is one of the classical hallmarks of AD and proposed as a critical driver of AD pathogenesis (amyloid cascade hypothesis). We identified transcripts spatially localized to Aβ pathology conserved between both human and mouse. We also integrated spatial and single-nucleus transcriptomic data to discover spatially defined cell signaling pathways dysregulated with disease and highlight regional heterogeneity of astrocytes. Altogether, this dissertation reveals regional and cellular molecular changes occurring in AD and contextualizes them in a systems-level framework to uncover pathways for further study in AD

Systems biology approaches to unravel the molecular and genetic architecture of Alzheimer's disease and related tauopathies

Over the years, genetic studies have identified multiple genetic risk variants associated with neurodegenerative disorders and helped reveal new biological pathways and genes of interest. However, genetic risk variants commonly reside in non-coding regions and may regulate distant genes rather than the nearest gene, as well as a gene's interaction partners in biological networks. Systems biology and functional genomics approaches provide the framework to unravel the functional significance of genetic risk variants in disease. In this review, we summarize the genetic and transcriptomic studies of Alzheimer's disease and related tauopathies and focus on the advantages of performing systems-level analyses to interrogate the biological pathways underlying neurodegeneration. Finally, we highlight new avenues of multi-omics analysis with single-cell approaches, which provide unparalleled opportunities to systematically explore cellular heterogeneity, and present an example of how to integrate publicly available single-cell datasets. Systems-level analysis has illuminated the function of many disease risk genes, but much work remains to study tauopathies and to understand spatiotemporal gene expression changes of specific cell types

Protocol for single-nucleus ATAC sequencing and bioinformatic analysis in frozen human brain tissue.

Single-nucleus ATAC sequencing (snATAC-seq) employs a hyperactive Tn5 transposase to gain precise information about the cis-regulatory elements in specific cell types. However, the standard protocol of snATAC-seq is not optimized for all tissues, including the brain. Here, we present a modified protocol for single-nuclei isolation from postmortem frozen human brain tissue, followed by snATAC-seq library preparation and sequencing. We also describe an integrated bioinformatics analysis pipeline using an R package (ArchRtoSignac) to robustly analyze snATAC-seq data. For complete details on the use and execution of this protocol, please refer to Morabito et al. (2021)

Adjunctive Therapies to Cerclage for the Prevention of Preterm Birth: A Systematic Review

The aim of this paper is to provide a thorough summary of published studies that have assessed the efficacy of adjunctive therapies used in addition to cervical cerclage as a preventive measure for preterm birth. We limited our paper to patients treated with cerclage plus an additional prophylactic therapy compared to a reference group of women with cerclage alone. The specific adjunctive therapies included in this systematic review are progesterone, reinforcing or second cerclage placement, tocolytics, antibiotics, bedrest, and pessary. We searched PubMed and Cochrane databases without date criteria with restriction to English language and human studies and performed additional bibliographic review of selected articles and identified 305 total studies for review. Of those, only 12 studies compared the use of an adjunctive therapy with cerclage to a reference group of cerclage alone. None of the 12 were prospective randomized clinical trials. No comparative studies were identified addressing the issues of antibiotics, bedrest, or pessary as adjunctive treatments to cerclage. None of the 12 studies included in this paper demonstrated a clear benefit of any adjunctive therapy used in addition to cerclage over and above cerclage used alone; however, few studies with small numbers limited the strength of the conclusions