Biology subjects

Nowakowski, T. J.

Publications and source records attributed to Nowakowski, T. J..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Adhesion G protein-coupled receptor ADGRG1 promotes protective microglial response in Alzheimer's disease

Germline genetic architecture of Alzheimers disease (AD) indicates microglial mechanisms of disease susceptibility and outcomes. However, the mechanisms that enable microglia to mediate protective responses to AD pathology remain elusive. Adgrg1 is specifically expressed in yolk-sac-derived microglia. This study reveals the role of yolk-sac-derived microglia in AD pathology, highlighting the function of ADGRG1 in modulating microglial protective responses to amyloid deposition. Utilizing both constitutive and inducible microglial Adgrg1 knockout 5xFAD models, we demonstrate that Adgrg1 deficiency leads to increased amyloid deposition, exacerbated neuropathology, and accelerated cognitive impairment. Transcriptomic analyses reveal a distinct microglial state characterized by downregulated genes associated with homeostasis, phagocytosis, and lysosomal functions. Functional assays in mouse models and human embryonic stem cells-derived microglia support that microglial ADGRG1 is required for efficient A{beta} phagocytosis. Together, these results uncover a GPCR-dependent microglial response to A{beta}, pointing towards potential therapeutic strategies to alleviate disease progression by enhancing microglial functional competence.

neuroscience↗

bioRxiv2024

High-Complexity Barcoded Rabies Virus for Scalable Circuit Mapping Using Single-Cell and Single-Nucleus Sequencing

Single cell genomics has revolutionized our understanding of neuronal cell types. However, scalable technologies for probing single-cell connectivity are lacking, and we are just beginning to understand how molecularly defined cell types are organized into functional circuits. Here, we describe a protocol to generate high-complexity barcoded rabies virus (RV) for scalable circuit mapping from tens of thousands of individual starter cells in parallel. In addition, we introduce a strategy for targeting RV-encoded barcode transcripts to the nucleus so that they can be read out using single-nucleus RNA sequencing (snRNA-seq). We apply this tool in organotypic slice cultures of the developing human cerebral cortex, which reveals the emergence of cell type- specific circuit motifs in midgestation. By leveraging the power and throughput of single cell genomics for mapping synaptic connectivity, we chart a path forward for scalable circuit mapping of molecularly-defined cell types in healthy and disease states.

neuroscience↗

bioRxiv2024

Single-cell multidimensional profiling of tumor cell heterogeneity in supratentorial ependymomas

Supratentorial ependymomas are aggressive childhood brain cancers that retain features of neurodevelopmental cell types and segregate into molecularly and clinically distinct subgroups, suggesting different developmental roots. The developmental signatures as well as microenvironmental factors underlying aberrant cellular transformation and behavior across each supratentorial ependymoma subgroup are unknown. Here we integrated single cell- and spatial transcriptomics, as well as in vitro and in vivo live-cell imaging to define supratentorial ependymoma cell states, spatial organization, and dynamic behavior within the neural microenvironment. We find that individual tumor subgroups harbor two distinct progenitor-like cell states reminiscent of early human brain development and diverge in the extent of neuronal or ependymal differentiation. We further uncover several modes of spatial organization of these tumors, including a high order architecture influenced by mesenchymal and hypoxia signatures. Finally, we identify an unappreciated role for brain-resident cells in shifting supratentorial ependymoma cellular heterogeneity towards neuronal-like cells that co-opt immature neuronal morphology and invasion mechanisms. Collectively, these findings provide a multidimensional framework to integrate transcriptional and phenotypic characterization of tumor heterogeneity in supratentorial ependymoma and its potential clinical implications.

cancer biology↗

bioRxiv2024

Ciliary biology intersects autism and congenital heart disease

Autism spectrum disorder (ASD) commonly co-occurs with congenital heart disease (CHD), but the molecular mechanisms underlying this comorbidity remain unknown. Given that children with CHD come to clinical attention by the newborn period, understanding which CHD variants carry ASD risk could provide an opportunity to identify and treat individuals at high risk for developing ASD far before the typical age of diagnosis. Therefore, it is critical to delineate the subset of CHD genes most likely to increase the risk of ASD. However, to date there is relatively limited overlap between high confidence ASD and CHD genes, suggesting that alternative strategies for prioritizing CHD genes are necessary. Recent studies have shown that ASD gene perturbations commonly dysregulate neural progenitor cell (NPC) biology. Thus, we hypothesized that CHD genes that disrupt neurogenesis are more likely to carry risk for ASD. Hence, we performed an in vitro pooled CRISPR interference (CRISPRi) screen to identify CHD genes that disrupt NPC biology similarly to ASD genes. Overall, we identified 45 CHD genes that strongly impact proliferation and/or survival of NPCs. Moreover, we observed that a cluster of physically interacting ASD and CHD genes are enriched for ciliary biology. Studying seven of these genes with evidence of shared risk (CEP290, CHD4, KMT2E, NSD1, OFD1, RFX3, TAOK1), we observe that perturbation significantly impacts primary cilia formation in vitro. While in vivo investigation of TAOK1 reveals a previously unappreciated role for the gene in motile cilia formation and heart development, supporting its prediction as a CHD risk gene. Together, our findings highlight a set of CHD risk genes that may carry risk for ASD and underscore the role of cilia in shared ASD and CHD biology.

developmental biology↗

bioRxiv2024

CRISPRi-based screen of Autism Spectrum Disorder risk genes in microglia uncovers roles of ADNP in microglia endocytosis and uptake of synaptic material

Autism Spectrum Disorders (ASD) are a set of neurodevelopmental disorders with complex biology. The identification of ASD risk genes from exome-wide association studies and de novo variation analyses has enabled mechanistic investigations into how ASD-risk genes alter development. Most functional genomics studies have focused on the role of these genes in neurons and neural progenitor cells. However, roles for ASD risk genes in other cell types are largely uncharacterized. There is evidence from postmortem tissue that microglia, the resident immune cells of the brain, appear activated in ASD. Here, we used CRISPRi-based functional genomics to systematically assess the impact of ASD risk gene knockdown on microglia activation and phagocytosis. We developed an iPSC-derived microglia-neuron coculture system and high-throughput flow cytometry readout for synaptic pruning to enable parallel CRISPRi-based screening of phagocytosis of beads, synaptosomes, and synaptic pruning. Our screen identified ADNP, a high-confidence ASD risk genes, as a modifier of microglial synaptic pruning. We found that microglia with ADNP loss have altered endocytic trafficking, remodeled proteomes, and increased motility in coculture.

neuroscience↗

bioRxiv2023

A foundational atlas of autism protein interactions reveals molecular convergence

Translating high-confidence (hc) autism spectrum disorder (ASD) genes into viable treatment targets remains elusive. We constructed a foundational protein-protein interaction (PPI) network in HEK293T cells involving 100 hcASD risk genes, revealing over 1,800 PPIs (87% novel). Interactors, expressed in the human brain and enriched for ASD but not schizophrenia genetic risk, converged on protein complexes involved in neurogenesis, tubulin biology, transcriptional regulation, and chromatin modification. A PPI map of 54 patient-derived missense variants identified differential physical interactions, and we leveraged AlphaFold-Multimer predictions to prioritize direct PPIs and specific variants for interrogation in Xenopus tropicalis and human forebrain organoids. A mutation in the transcription factor FOXP1 led to reconfiguration of DNA binding sites and altered development of deep cortical layer neurons in forebrain organoids. This work offers new insights into molecular mechanisms underlying ASD and describes a powerful platform to develop and test therapeutic strategies for many genetically-defined conditions.

systems biology↗

bioRxiv2023

Inhibition of muscarinic receptor signaling protects human enteric inhibitory neurons against platin chemotherapy toxicity

GI toxicity is a common dose-limiting adverse effect of platin chemotherapy treatment. Up to 50% of cancer survivors continue to experience symptoms of chronic constipation or diarrhea induced by their chemotherapy for many years after their treatment. This drug toxicity is largely attributed to damage to enteric neurons that innervate the GI tract and control GI motility. The mechanisms responsible for platin-induced enteric neurotoxicity and potential preventative strategies have remained unknown. Here, we use human pluripotent stem cell derived enteric neurons to establish a new model system capable of uncovering the mechanism of platin-induced enteric neuropathy. Utilizing this scalable system, we performed a high throughput screen and identified drug candidates and pathways involved in the disease. Our analyses revealed that excitotoxicity through muscarinic cholinergic signaling is a key driver of platin-induced enteric neuropathy. Using single nuclei transcriptomics and functional assays, we discovered that this disease mechanism leads to increased susceptibility of specific neuronal subtypes, including inhibitory nitrergic neurons, to platins. Histological assessment of the enteric nervous system in platin-treated patients confirmed the selective loss of nitrergic neurons. Finally, we demonstrated that pharmacological and genetic inhibition of muscarinic cholinergic signaling is sufficient to rescue enteric neurons from platin excitotoxicity in vitro and can prevent platin-induced constipation and degeneration of nitrergic neurons in mice. These studies define the mechanisms of platin-induced enteric neuropathy and serve as a framework for uncovering cell type-specific manifestations of cellular stress underlying numerous intractable peripheral neuropathies.

pharmacology and toxicology↗

bioRxiv2023

Massively parallel characterization of psychiatric disorder-associated and cell-type-specific regulatory elements in the developing human cortex

Nucleotide changes in gene regulatory elements are important determinants of neuronal development and disease. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 sequences, including differentially accessible cell-type specific regions in the developing cortex and single-nucleotide variants associated with psychiatric disorders. In primary cells, we identified 46,802 active enhancer sequences and 164 disorder-associated variants that significantly alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning, we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development. One Sentence SummaryWe identify 46,802 enhancers and 164 psychiatric disorder variants with regulatory effects in the developing cortex and organoids.

genomics↗

bioRxiv2022

Single-cell analysis of prenatal and postnatal human cortical development

We analyze more than 700,000 single-nucleus RNA-seq profiles from 106 donors during prenatal and postnatal developmental stages and identify lineage-specific programs that underlie the development of specific subtypes of excitatory cortical neurons, interneurons, glial cell types and brain vasculature. By leveraging single-nucleus chromatin accessibility data, we delineate enhancer-gene regulatory networks and transcription factors that control commitment of specific cortical lineages. By intersecting our results with genetic risk factors for human brain diseases, we identify the cortical cell types and lineages most vulnerable to genetic insults of different brain disorders, especially autism. We find that lineage-specific gene expression programs upregulated in female cells are especially enriched for the genetic risk factors of autism. Our study captures the molecular progression of cortical lineages across human development. One Sentence SummarySingle-cell transcriptomic atlas of human cortical development identifies lineage and sex-specific programs and their implication in brain disorders.

neuroscience↗

bioRxiv2022

Rubella virus tropism and single cell responses in human primary tissue and microglia-containing organoids

Rubella virus is an important human pathogen that can cause neurologic deficits in a developing fetus when contracted during pregnancy. Despite successful vaccination programs in the Americas and many developed countries, rubella remains endemic in many regions worldwide and outbreaks occur wherever population immunity is insufficient. Intense interest since rubella virus was first isolated in 1962 has advanced our understanding of clinical outcomes after infection disrupts key processes of fetal neurodevelopment. Yet it is still largely unknown which cell types in the developing brain are targeted. We show that in human brain slices, rubella virus predominantly infects microglia. This infection occurs in a heterogeneous population but not in a highly microglia-enriched monoculture in the absence of other cell types. By using an organoid-microglia model, we further demonstrate that rubella virus infection leads to a profound interferon response in non-microglial cells, including neurons and neural progenitor cells, and this response is attenuated by the presence of microglia.

developmental biology↗

bioRxiv2022

Epigenomic and chromosomal architectural reconfiguration in developing human frontal cortex and hippocampus

The human frontal cortex and hippocampus play critical roles in learning and cognition. We investigated the epigenomic and 3D chromatin conformational reorganization during the development of the frontal cortex and hippocampus, using more than 53,000 joint single-nucleus profiles of chromatin conformation and DNA methylation (sn-m3C-seq). The remodeling of DNA methylation predominantly occurs during late-gestational to early-infant development and is temporally separated from chromatin conformation dynamics. Neurons have a unique Domain-Dominant chromatin conformation that is different from the Compartment-Dominant conformation of glial cells and non-brain tissues. We reconstructed the regulatory programs of cell-type differentiation and found putatively causal common variants for schizophrenia strongly overlap with chromatin loop-connected, cell-type-specific regulatory regions. Our data demonstrate that single-cell 3D-regulome is an effective approach for dissecting neuropsychiatric risk loci.

genomics↗

bioRxiv2022

Three-dimensional genome re-wiring in loci with Human Accelerated Regions

Human Accelerated Regions (HARs) are conserved genomic loci that evolved at an accelerated rate in the human lineage and may underlie human-specific traits. We generated HARs and chimpanzee accelerated regions with the largest alignment of mammalian genomes to date. To facilitate exploration of accelerated evolution in other lineages, we implemented an open-source Nextflow pipeline that runs on any computing platform. Combining deep-learning with chromatin capture experiments in human and chimpanzee neural progenitor cells, we discovered a significant enrichment of HARs in topologically associating domains (TADs) containing human-specific genomic variants that change three-dimensional (3D) genome organization. Differential gene expression between humans and chimpanzees at these loci in multiple cell types suggests rewiring of regulatory interactions between HARs and neurodevelopmental genes. Thus, comparative genomics together with models of 3D genome folding revealed enhancer hijacking as an explanation for the rapid evolution of HARs. One-Sentence SummaryHuman-specific changes to 3D genome organization may have contributed to rapid evolution of mammalian-conserved loci in the human genome.

evolutionary biology↗

bioRxiv2022

Transcriptomic cell type structures in vivo neuronal activity across multiple time scales.

SUMMARYCell type is hypothesized to be a key determinant of the role of a neuron within a circuit. However, it is unknown whether a neurons transcriptomic type influences the timing of its activity in the intact brain. In other words, can transcriptomic cell type be extracted from the time series of a neurons activity? To address this question, we developed a new deep learning architecture that learns features of interevent intervals across multiple timescales (milliseconds to >30 min). We show that transcriptomic cell class information is robustly embedded in the timing of single neuron activity recorded in the intact brain of behaving animals (calcium imaging and extracellular electrophysiology), as well as in a bio-realistic model of visual cortex. In contrast, we were unable to reliably extract cell identity from summary measures of rate, variance, and interevent interval statistics. We applied our analyses to the question of whether transcriptomic subtypes of excitatory neurons represent functionally distinct classes. In the calcium imaging dataset, which contains a diverse set of excitatory Cre lines, we found that a subset of excitatory cell types are computationally distinguishable based upon their Cre lines, and that excitatory types can be classified with higher accuracy when considering their cortical layer and projection class. Here we address the fundamental question of whether a neuron, within a complex cortical network, embeds a fingerprint of its transcriptomic identity into its activity. Our results reveal robust computational fingerprints for transcriptomic types and classes across diverse contexts, defined over multiple timescales.

neuroscience↗

bioRxiv2022

hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function

The enteric nervous system (ENS) plays a central role in gut physiology and mediating the crosstalk between the gastrointestinal (GI) tract and other organs. The human ENS has remained elusive, highlighting the need for an in vitro modeling and mapping blueprint. Here we map out the developmental and functional features of the human ENS, by establishing robust and scalable 2D ENS cultures and 3D enteric ganglioids from human pluripotent stem cells (hPSCs). These models recapitulate the remarkable neuronal and glial diversity found in primary tissue and enable comprehensive molecular analyses that uncover functional and developmental relationships within these lineages. As a salient example of the power of this system, we performed in-depth characterization of enteric nitrergic neurons (NO neurons) which are implicated in a wide range of GI motility disorders. We conducted an unbiased screen and identified drug candidates that modulate the activity of NO neurons and demonstrated their potential in promoting motility in mouse colonic tissue ex vivo. We established a high-throughput strategy to define the developmental programs involved in NO neuron specification and discovered that PDGFR inhibition boosts the induction of NO neurons in enteric ganglioids. Transplantation of these ganglioids in the colon of NO neuron-deficient mice results in extensive tissue engraftment, providing a xenograft model for the study of human ENS in vivo and the development of cell-based therapies for neurodegenerative GI disorders. These studies provide a framework for deciphering fundamental features of the human ENS and designing effective strategies to treat enteric neuropathies.

developmental biology↗

bioRxiv2021

Machine learning-based library design improves packaging and diversity of adeno-associated virus (AAV) libraries

Adeno-associated viruses (AAVs) hold tremendous promise as delivery vectors for clinical gene therapy, but they need improvement. AAVs with enhanced properties, such as more efficient and/or cell-type specific infection, can be engineered by creating a large, diverse starting library and screening for desired phenotypes, in some cases iteratively. Although this approach has succeeded in numerous specific cases, such as infecting cell types from the brain to the lung, the starting libraries often contain a high proportion of variants unable to assemble or package their genomes, a general prerequisite for engineering any gene delivery goal. Herein, we develop and showcase a machine learning (ML)-based method for systematically designing more effective starting libraries -- ones that have broadly good packaging capabilities while being as diverse as possible. Such carefully designed but general libraries stand to significantly increase the chance of success in engineering any property of interest. Furthermore, we use this approach to design a clinically-relevant AAV peptide insertion library that achieves 5-fold higher packaging fitness than the state-of-the-art library, with negligible reduction in diversity. We demonstrate the general utility of this designed library on a downstream task to which our approach was agnostic: infection of primary human brain tissue. The ML-designed library had approximately 10-fold more successful variants than the current state-of-the-art library. Not only should our new library serve useful for any number of other engineering goals, but our library design approach itself can also be applied to other types of libraries for AAV and beyond.

bioengineering↗

bioRxiv2021

Integrated gene analyses of de novo mutations from 46,612 trios with autism and developmental disorders

Most genetic studies consider autism spectrum disorder (ASD) and developmental disorder (DD) separately despite overwhelming comorbidity and shared genetic etiology. Here we analyzed de novo mutations (DNMs) from 15,560 ASD (6,557 are new) and 31,052 DD trios independently and combined as broader neurodevelopmental disorders (NDD) using three models. We identify 615 candidate genes (FDR 5%, 189 potentially novel) by one or more models, including 138 reaching exome-wide significance (p < 3.64e-07) in all models. We find no evidence for ASD-specific genes in contrast to 18 genes significantly enriched for DD. There are 53 genes show particular mutational-bias including enrichments for missense (n=41) or truncating DNM (n=12). We find 22 genes with evidence of sex-bias including five X chromosome genes also with significant female burden (DDX3X, MECP2, SMC1A, WDR45, and HDAC8). NDD risk genes group into five functional networks associating with different brain developmental lineages based on single-cell nuclei transcriptomic data, which provides important insights into disease subtypes and future functional studies.

genetics↗

bioRxiv2021

Internet of Things Architecture for High Throughput Biology

The Internet of Things (IoT) provides a simple framework to easily control online devices. IoT is now a commonplace tool used by technology companies, but it is rarely used in biology experiments. IoT can benefit research through alarm notifications, automation, and the real-time monitoring of experiments. We developed and implemented an IoT architecture to control biological devices used in experiments. We developed our own electrophysiology, microscopy, and microfluidic devices so that may be controlled through a unified IoT architecture. The system allows each device to be monitored and controlled through an online web tool. We present our IoT architecture so other labs may replicate it for their own experiments.

bioengineering↗

bioRxiv2021

Light-weight Electrophysiology Hardware and Software Platform for Cloud-Based Neural Recording Experiments

ObjectiveNeural activity represents a functional readout of neurons that is increasingly important to monitor in a wide range of experiments. Extracellular recordings have emerged as a powerful technique for measuring neural activity because these methods do not lead to the destruction or degradation of the cells being measured. Current approaches to electrophysiology have a low throughput of experiments due to manual supervision and expensive equipment. This bottleneck limits broader inferences that can be achieved with numerous long-term recorded samples. ApproachWe developed Piphys, an inexpensive open source neurophysiological recording platform that consists both hardware and software. It is easily accessed and controlled via a standard web interface through Internet of Things (IoT) protocols. Main ResultsWe used a Raspberry Pi as the primary processing device and Intan bioamplifier. We designed a hardware expansion circuit board and software to enable voltage sampling and user interaction. This standalone system was validated with primary human neurons, showing reliability in collecting real-time neural activity. SignificanceThe hardware modules and cloud software allow for remote control of neural recording experiments as well as horizontal scalability, enabling long-term observations of development, organization, and neural activity at scale.

bioengineering↗