Biology subjects

Milbrandt, J.

Publications and source records attributed to Milbrandt, J..

24 records · Page 2Linked to original sources

bioRxiv2021

Measuring Transcription Factor Binding and Gene Expression using Barcoded Self-Reporting Transposon Calling Cards and Transcriptomes

Calling cards technology using self-reporting transposons enables the identification of DNA-protein interactions through RNA sequencing. Although immensely powerful, current implementations of calling cards in bulk experiments on populations of cells are technically cumbersome and require many replicates to identify independent insertions into the same genomic locus. Here, we have drastically reduced the cost and labor requirements of calling card experiments in bulk populations of cells by introducing a DNA barcode into the calling card itself. An additional barcode incorporated during reverse transcription enables simultaneous transcriptome measurement in a facile and affordable protocol. We demonstrate that barcoded self-reporting transposons recover in vitro binding sites for four basic helix-loop-helix transcription factors with important roles in cell fate specification: ASCL1, MYOD1, NEUROD2, and NGN1. Further, simultaneous calling cards and transcriptional profiling during transcription factor overexpression identified both binding sites and gene expression changes for two of these factors. Lastly, we demonstrated barcoded calling cards can record binding in vivo in the mouse brain. In sum, RNA-based identification of transcription factor binding sites and gene expression through barcoded self-reporting transposon calling cards and transcriptomes is an efficient and powerful method to infer gene regulatory networks in a population of cells.

genomics↗

bioRxiv2021

Activation of Sarm1 produces cADPR to increase intra-axonal calcium and promote axon degeneration in CIPN

Cancer patients frequently develop chemotherapy-induced peripheral neuropathy (CIPN), a painful and long-lasting disorder with profound somatosensory deficits. There are no effective therapies to prevent or treat this disorder. Pathologically, CIPN is characterized by a "dying-back" axonopathy that begins at intra-epidermal nerve terminals of sensory neurons and progresses in a retrograde fashion. Calcium dysregulation constitutes a critical event in CIPN, but it is not known how chemotherapies such as paclitaxel alter intra-axonal calcium and cause degeneration. Here, we demonstrate that paclitaxel triggers Sarm1-dependent cADPR production in distal axons, promoting intra-axonal calcium flux from both intracellular and extracellular calcium stores. Genetic or pharmacologic antagonists of cADPR signaling prevent paclitaxel-induced axon degeneration and allodynia symptoms, without mitigating the anti-neoplastic efficacy of paclitaxel. Our data demonstrate that cADPR is a calcium modulating factor that promotes paclitaxel-induced axon degeneration and suggest that targeting cADPR signaling provides a potential therapeutic approach for treating CIPN. HIGHLIGHTSO_LIPaclitaxel induces intra-axonal calcium flux C_LIO_LISarm1-dependent cADPR production promotes axonal calcium elevation and degeneration C_LIO_LIAntagonizing cADPR signaling pathway protects against paclitaxel-induced peripheral neuropathy in vitro and in vivo C_LI

neuroscience↗

bioRxiv2020

SARM1 depletion rescues NMNAT1 dependent photoreceptor cell death and retinal degeneration.

Leber congenital amaurosis type 9 is an autosomal recessive retinopathy caused by mutations of the NAD+ synthesis enzyme NMNAT1. Despite the ubiquitous expression of NMNAT1, patients do not manifest pathologies other than retinal degeneration. Here we demonstrate that widespread NMNAT1 depletion in adult mice mirrors the human pathology, with selective loss of photoreceptors highlighting the exquisite vulnerability of these cells to NMNAT1 loss. Conditional deletion demonstrates that NMNAT1 is required within the photoreceptor. Mechanistically, loss of NMNAT1 activates the NADase SARM1, the central executioner of axon degeneration, to trigger photoreceptor death and vision loss. Hence, the essential function of NMNAT1 in photoreceptors is to inhibit SARM1, highlighting an unexpected shared mechanism between axonal degeneration and photoreceptor neurodegeneration. These results define a novel SARM1-dependent photoreceptor cell death pathway and identifies SARM1 as a therapeutic candidate for retinopathies.

neuroscience↗

bioRxiv2019

Peripheral nerve resident macrophages are microglia-like cells with tissue-specific programming

Whereas microglia are recognized as fundamental players in central nervous system (CNS) development and function, much less is known about macrophages of the peripheral nervous system (PNS). Here we show that self-maintaining PNS macrophages share unique features with CNS microglia. By comparing gene expression across neural and conventional tissue-resident macrophages, we identified transcripts that were shared among neural resident macrophages as well as selectively enriched in PNS macrophages. Remarkably, PNS macrophages constitutively expressed genes previously identified to be upregulated by activated microglia during aging or neurodegeneration. Several microglial activation-associated and PNS macrophage-enriched genes were also expressed in spinal cord microglia at steady state. While PNS macrophages arose from both embryonic and hematopoietic precursors, their expression of activation-associated genes did not differ by ontogeny. Collectively, these data uncover shared and unique features between neural resident macrophages and emphasize the role of nerve environment for shaping PNS macrophage identity.

immunology↗

bioRxiv2019

Multiplexed single-cell autism modeling reveals convergent mechanisms altering neuronal differentiation

The overwhelming success of exome- and genome-wide association studies in discovering thousands of disease-associated genes necessitates novel high-throughput functional genomics approaches to elucidate the mechanisms of these genes. Here, we have coupled multiplexed repression of neurodevelopmental disease-associated genes to single-cell transcriptional profiling in differentiating human neurons to rapidly assay the functions of multiple genes in a disease-relevant context, assess potentially convergent mechanisms, and prioritize genes for specific functional assays. For a set of 13 autism spectrum disorder (ASD) associated genes, we demonstrate that this approach generated important mechanistic insights, revealing two functionally convergent modules of ASD genes: one that delays neuron differentiation and one that accelerates it. Five genes that delay neuron differentiation (ADNP, ARID1B, ASH1L, CHD2, and DYRK1A) mechanistically converge, as they all dysregulate genes involved in cell-cycle control and progenitor cell proliferation. Live-cell imaging after individual ASD gene repression validated this functional module, confirming that these genes reduce neural progenitor cell proliferation and neurite growth. Finally, these functionally convergent ASD gene modules predicted shared clinical phenotypes among individuals with mutations in these genes. Altogether these results demonstrate the utility of a novel and simple approach for the rapid functional elucidation of neurodevelopmental disease-associated genes.

genomics↗

bioRxiv2018

Single cell RNAseq uncovers a robust transcriptional response to morphine by oligodendrocytes

Molecular and behavioral responses to opioids are thought to be primarily mediated by neurons, although there is accumulating evidence that other cell types also play a role in drug addiction. To investigate cell-type-specific opioid responses, we performed single-cell RNA sequencing of the nucleus accumbens of mice following acute morphine treatment. Differential expression analysis uncovered robust morphine-dependent changes in gene expression in oligodendrocytes. We examined the expression of selected genes, including Cdkn1a and Sgk1, by FISH, confirming their induction by morphine in oligodendrocytes. Further analysis using RNAseq of FACS-purified oligodendrocytes revealed a large cohort of morphine-regulated genes. Importantly, the affected genes are enriched for roles in cellular pathways intimately linked to oligodendrocyte maturation and myelination, including the unfolded protein response. Altogether, our data shed light on a novel, morphine-dependent transcriptional response by oligodendrocytes that may contribute to the myelination defects observed in human opioid addicts.

neuroscience↗