Biology subjects

Smith, B. M.

Publications and source records attributed to Smith, B. M..

15 recordsLinked to original sources

bioRxiv2026

Classic and novel features of the dentate gyrus vascular niche quantified through 3D vessel reconstruction

Adult neurogenesis, the continued generation of new neurons throughout life, supports a variety of emotional and cognitive functions in mammals. Lifelong neurogenesis occurs in the dentate gyrus (DG) of the hippocampus, where a neurogenic niche supports the survival and growth of neural stem cells (NSCs), which divide to generate new neurons. A key component of this niche is the surrounding vasculature, thought to give NSCs access to circulating support molecules, oxygen, and nutrient metabolites. The DG vasculature is strikingly variable across layers. Vascular density and linearity, for example, have been noted to differ substantially in the different cell layers that make up the DG. Most of these prior descriptions of the DG vasculature have been qualitative, and other important aspects of vessel morphology, like vessel diameter, have not been previously reported to our knowledge. To provide a more complete and quantitative description of the adult DG vasculature, we used a 3D imaging technique to fully characterize the vasculature of each DG cellular layer. Our 3D reconstructions of DG blood vessels provide a quantitative method for measuring several known, qualitative features of the neurogenic niche, such as blood vessel density and tortuosity. In some cases, we reproduce qualitative observations, such as affirming that vessel density is high in the subgranular zone (SGZ) where NSCs reside and low in the granule cell layer. In other cases, our findings go counter to what qualitative observations suggest. For example, the SGZ is often described as having uniquely linear blood vessels, but we found that linearity was similar between the SGZ and hilus layer. We also report new measures of features of DG layer vascular architecture: vessel diameter and branching. Together, these findings offer a global, quantitative characterization of the DG vascular niche in adult mice. Beyond providing more detailed characterization of DG vasculature, we also anticipate our methods and estimates of variance in mice will be useful for investigators wishing to plan their own quantitative studies of changes in the vascular structure of the DG.

neuroscience↗

bioRxiv2023

Efficient Memory Encoding Explains the Interactions Between Hippocampus Size, Individual Experience, and Clinical Outcomes: A Computational Model

The relationship between hippocampal volume and memory function has produced mixed results in neuroscience research, which suggests an additional influencing mechanism. To explore the role of an experience-dependent encoding mechanism, we developed an autoencoder model of the cortex-hippocampus loop and examined how its memory representation is affected by a penalty that prioritizes sparseness. We trained our model with the Fashion MNIST database and a loss function to modify synapses via backpropagation of mean squared recall error. The model exhibited experience-dependent efficient encoding, representing frequently-repeated objects with fewer neurons and smaller loss penalties; representations of similar sizes were found for objects repeated equally. Our findings clarify perplexing results from neurodevelopmental studies by linking increased hippocampal size and memory impairments in autism spectrum disorder (ASD) to decreased sparseness, and explaining dementia symptoms of forgetting with varied neuronal integrity. Our findings propose a novel model that connects observed relationships between hippocampal size and memory to the environmental demands and the underlying biological constraints, contributing to the development of a larger theory on experience-dependent encoding and storage and its failure. Author SummaryThe hippocampus is the brain region where memories are initially formed. A larger hippocampus seems to be associated with better memory performance, but this is not a static relationship; studies suggest that its size might grows with demand (e.g., the need to memorize more information) and that, in certain conditions, larger or smaller volumes of the hippocampus are not associated with better or worse memory. We demonstrate that it is possible to make sense of these findings by assuming that the hippocampus balances the need to correctly remember with the need to minimize resources used to store it, and that the hippocampal size might change as a function of memory demands (the characteristics of the information to memorize) and the underlying biology (e.g., the presence of hippocampal cell damage, or a reduction in hippocampal GABA receptors). We test this hypothesis by building a neural network model; the model correctly predicts existing, puzzling findings in the literature, and can even capture subtle interactions between different phenomena, such as predicting that individuals with Autism Spectrum Disorder would be more susceptible to memory loss in dementia.

neuroscience↗

bioRxiv2022

Individual differences in volitional social motivation in male and female mice following social stress.

BackgroundA key challenge in developing new treatments for neuropsychiatric illness is the disconnect between preclinical models and the complexity of human social behavior. We aimed to integrate voluntary social self-administration into a preclinical rodent stress model, as a platform for the identification of basic brain and behavior mechanisms underlying stress-induced individual differences in social motivation. Here, we introduce an operant social stress (OSS) procedure with male and female mice, where lever presses are reinforced by freely moving social interaction with a familiar social partner across social stress exposure. MethodsOSS is composed of three phases: (i) social self-administration training, (ii) social stress concurrent with daily reinforced social self-administration testing, and (iii) post-stress operant social reward testing under both non-reinforced and reinforced conditions. We resolve social stress-induced changes to social motivation behaviors using hierarchical clustering and aggregated z-scores, capturing the spectrum of individual differences that we describe with a social index score. ResultsOSS captures a range of stress-related dynamic social motivation behaviors inclusive of sex as a biological variable. Both male and female mice lever press for access to a social partner, independent of social partner coat color or familiarity. Social stress attenuates social self-administration in males and promotes social reward seeking behavior in females. Hierarchical clustering does not adequately describe the relative distributions of social motivation following stress, which we find is better described as a non-binary behavioral distribution that we define by introducing the social index score. This index is stable across individual mice. ConclusionWe demonstrate that OSS can be used to detect stable individual differences in stress-induced changes to social motivation in male and female mice. These differences may reflect unique neurobiological, cellular and circuit mechanisms not captured by preclinical models that omit voluntary social behaviors. The inclusion of volitional social procedures may enhance the understanding of behavioral adaptations promoting stress resiliency and their mechanisms under more naturalistic conditions.

neuroscience↗

bioRxiv2022

Dual Targeting of FLT3 and LSD1 Disrupts the MYC Super-Enhancer Complex in Acute Myeloid Leukemia

Mutations in Fms-like tyrosine kinase 3 (FLT3) are common drivers in acute myeloid leukemia (AML) yet FLT3 inhibitors only provide modest clinical benefit. Prior work has shown that inhibitors of lysine-specific demethylase 1 (LSD1) enhance kinase inhibitor activity in AML. Here we show that combined LSD1 and FLT3 inhibition induces synergistic cell death in FLT3-mutant AML. Multi-omic profiling revealed that the drug combination disrupts STAT5, LSD1, and GFI1 binding at the MYC blood super-enhancer, suppressing super-enhancer activation as well as MYC expression and activity. The drug combination simultaneously results in the accumulation of repressive H3K9me1 methylation, an LSD1 substrate, at MYC target genes. We validated these findings in 72 primary AML samples with the nearly every sample demonstrating synergistic responses to the drug combination. Collectively, these studies provide preclinical rationale for the investigation of dual FLT3/LSD1 inhibition in a clinical trial.

cancer biology↗

bioRxiv2022

GoPeaks: Histone Modification Peak Calling for CUT&Tag

Genome-wide mapping of the histone modification landscape is critical to understanding tran-scriptional regulation. Cleavage Under Targets and Tagmentation (CUT&Tag) is a new method for profiling the localization of covalent histone modifications, offering improved sensitivity and decreased cost compared with Chromatin Immunoprecipitation Sequencing (ChIP-seq). Here, we present GoPeaks, a peak calling method specifically designed for histone modification CUT&Tag data. GoPeaks implements a Binomial distribution and stringent read count cut-off to nominate candidate genomic regions. We compared the performance of GoPeaks against commonly used peak calling algorithms to detect H3K4me3, H3K4me1, and H3K27Ac peaks from CUT&Tag data. These histone modifications display a range of peak profiles and are frequently used in epigenetic studies. We found GoPeaks robustly detects genome-wide histone modifications and, notably, identifies H3K27Ac with improved sensitivity compared to other standard peak calling algorithms.

bioinformatics↗

bioRxiv2022

Efficient generation of lower induced Motor Neurons by coupling Ngn2 expression with developmental cues

Human pluripotent stem cells (hPSCs) are a powerful tool for disease modelling and drug discovery, especially when access to primary tissue is limited, such as in the brain. Current neuronal differentiation approaches use either small molecules for directed differentiation or transcription-factor-mediated programming. In this study we coupled the overexpression of the neuralising transcription factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced Motor Neurons (liMoNes). We showed that this approach induced activation of the motor neuron (MN) specific transcription factor Hb9/MNX1, using anHb9::GFP-reporter line, with up to 95% of cells becoming Hb9::GFP+. These cells acquired and maintained expression of canonical early and mature MN markers. Molecular and functional profiling revealed that liMoNes resembled bona fide hPSC-derived MN differentiated by conventional small molecule patterning. liMoNes exhibited spontaneous electrical activity, expressed synaptic markers and formed contacts with muscle cells in vitro. Pooled, multiplex single-cell RNA sequencing on 50 cell lines revealed multiple anatomically distinct MN subtypes of cervical and brachial, limb-innervating MNs in reproducible quantities. We conclude that combining small molecule patterning with Ngn2 can facilitate the high-yield, robust and reproducible production of multiple disease-relevant MN subtypes, which is fundamental in the path to propel forward our knowledge of motoneuron biology and its disruption in disease.

neuroscience↗

bioRxiv2021

Mutant-SETBP1 activates transcription of Myc programs to accelerate CSF3R-driven myeloproliferative neoplasms

Colony stimulating factor 3 receptor (CSF3R) mutations lead to JAK pathway activation and are the molecular hallmark of chronic neutrophilic leukemia (CNL). Approximately half of CNL patients also have mutations in SET binding protein 1 (SETBP1). In this study, we developed models of SETBP1-mutant leukemia to understand the role that SETBP1 plays in CNL. SETBP1 mutations promote self-renewal of CSF3R-mutant hematopoietic progenitors in vitro and prevent cells from undergoing terminal differentiation. In vivo, SETBP1 mutations accelerate leukemia progression, leading to the rapid development of hepatosplenomegaly and granulocytosis. Through transcriptomic and epigenomic profiling, we found that SETBP1 enhances progenitor-associated programs--most strongly upregulating Myc and Myc target genes. This upregulation of Myc can be reversed by epigenetic modulatory drugs. In summary, we find that SETBP1 mutations promote aggressive hematopoietic cell expansion when expressed with mutant CSF3R through the upregulation of Myc-associated gene expression programs. Statement of SignificanceSETBP1 is frequently mutated in chronic neutrophilic leukemia, but its role in the biology of this disease is unclear. We find that mutant SETBP1 enhances transcription of Myc and Myc target genes to promote aggressive disease biology, and that these oncogenic transcriptional programs can be reversed by epigenetic modulatory drugs.

cancer biology↗

bioRxiv2021

Oral and injected tamoxifen alter adult hippocampal neurogenesis in female and male mice

Inducible Cre recombinase facilitates temporal control of genetic recombination in numerous transgenic model systems, a feature which has made it a popular tool for studies of adult neurogenesis. One of the most common forms of inducible Cre, CreERT2, requires activation by the synthetic estrogen tamoxifen (TAM) to initiate recombination of LoxP-flanked sequences. To date, most studies deliver TAM via intraperitoneal injection. But the introduction of TAM-infused commercial chows has recently expanded the possible modes of TAM delivery. Despite the widespread use of TAM-inducible genetic models in adult neurogenesis research, the comparative efficiency and off-target effects of TAM administration protocols is surprisingly infrequently studied. Here we compare a standard, 5 day TAM injection regimen with voluntary consumption of TAM-infused chow. First, we used adult NestinCreERT2;Rosa-LoxP-STOP-LoxP-EYFP reporter mice to show that 2 weeks of TAM chow and 5 days of injections led to LoxP recombination in a similar phenotypic population of neural stem and progenitor cells in the adult dentate gyrus. However, TAM chow resulted in substantially less overall recombination than injections. TAM administration also altered adult neurogenesis, but in different ways depending on administration route: TAM injection disrupted neural progenitor cell proliferation 3 weeks after TAM, whereas TAM chow increased neuronal differentiation of cells generated during the diet period. These findings provide guidance for selection of TAM administration route and appropriate controls in adult neurogenesis studies using TAM-inducible Cre mice. They also highlight the need for better understanding of off-target effects of TAM in other neurological processes and organ systems.

neuroscience↗

bioRxiv2021

PU.1 and MYC transcriptional network defines synergistic drug responses to KIT and LSD1 inhibition in acute myeloid leukemia

Activating mutations in the KIT tyrosine receptor kinase confer an adverse prognosis for patients with acute myeloid leukemia (AML). Outside of bone marrow transplantation, treatment options are limited. Here we demonstrate combined KIT and LSD1 inhibition produces synergistic cell death against KIT mutant AML cells. This combination suppresses MYC expression to drive cell cycle exit and apoptosis. This decreased MYC expression results from a loss of PU.1 binding at downstream MYC enhancers. The drug combination also inactivates PI3K/AKT/GSK3a/b signaling to decrease MYC protein abundance. KIT-mutant AML cells rapidly adapt to KIT inhibitor monotherapy by restoring PI3K/AKT activity, but cannot when treated with combined KIT and LSD1 inhibitor. In addition, we validate MYC suppression as a mechanism of synergy in KIT-mutant AML patient samples. Collectively, this work provides rational for a clinical trial to assess the efficacy of KIT and LSD1 inhibition in patients with KIT-mutant AML. Statement of significanceEffective treatment options for AML are limited. We describe the synergistic response to combined KIT and LSD1 inhibition in KIT-mutant AML and identify key biomarkers of drug response. The specificity and efficacy of this combination in cell lines and patient samples provides rationale for investigation in early phase clinical trials.

cancer biology↗

bioRxiv2021

Parallel and sequential pathways of molecular recognition of a tandem-repeat protein and its intrinsically disordered binding partner

The Wnt signalling pathway plays an important role in cell proliferation, differentiation and fate decisions in embryonic development and in the maintenance of adult tissues, and the twelve Armadillo (ARM) repeat-containing protein {beta}-catenin acts as the signal transducer in this pathway. Here we investigate the interaction between {beta}-catenin and the intrinsically disordered transcription factor TCF7L2, comprising a very long nanomolar-affinity interface of approximately 4800 [A]2 that spans ten of the twelve ARM repeats of {beta}-catenin. First, a fluorescence reporter system for the interaction was engineered and used to determine the kinetic rate constants for the association and dissociation. The association kinetics of TCF7L2 and {beta}-catenin was monophasic and rapid (7.3 {+/-} 0.1 x107 M-1s-1), whereas dissociation was biphasic and slow (5.7 {+/-} 0.4 x10-4 s-1, 15.2 {+/-} 2.8 x10-4 s-1). This reporter system was then combined with site-directed mutagenesis to investigate the striking variability in the conformation adopted by TCF7L2 in the three different crystal structures of the TCF7L2-{beta}-catenin complex. We found that mutation of the N- and C-terminal subdomains of TCF7L2 that adopt relatively fixed conformations in the crystal structures has a large effect on the dissociation kinetics, whereas mutation of the labile sub-domain connecting them has negligible effect. These results point to a two-site avidity mechanism of binding with the linker region forming a "fuzzy" complex involving transient contacts that are not site-specific. Strikingly, two mutations in the N-terminal subdomain that have the largest effects on the dissociation kinetics showed two additional phases, indicating partial flux through an alternative dissociation pathway that is inaccessible to the wild type. The results presented here provide insights into the kinetics of molecular recognition of a long intrinsically disordered region with an elongated repeat-protein surface, a process found to involve parallel routes with sequential steps in each.

biophysics↗

bioRxiv2021

Mutant SETBP1 enhances NRAS-driven MAPK pathway activation to promote aggressive leukemia

Mutations in SET binding protein 1 (SETBP1) are associated with poor outcomes in myeloid leukemias. In the Ras-driven leukemia, juvenile myelomonocytic leukemia, SETBP1 mutations are enriched in relapsed disease. While some mechanisms for SETBP1-driven oncogenesis have been established, it remains unclear how SETBP1 specifically modulates the biology of Ras-driven leukemias. In this study, we found that when co-expressed with Ras pathway mutations, SETBP1 promoted oncogenic transformation of murine bone marrow in vitro and aggressive myeloid leukemia in vivo. We demonstrate that SETBP1 enhances the NRAS gene expression signature, driving upregulation of mitogen-activated protein kinase (MAPK) signaling and downregulation of differentiation pathways. SETBP1 also enhances NRAS-driven phosphorylation of MAPK proteins. Cells expressing NRAS and SETBP1 are sensitive to inhibitors of the MAPK pathway, and treatment with the MEK inhibitor trametinib conferred a survival benefit in a mouse model of NRAS/SETBP1-mutant disease. Our data demonstrate that despite driving enhanced MAPK signaling, SETBP1-mutant cells remain susceptible to trametinib in vitro and in vivo, providing encouraging pre-clinical data for the use of trametinib in SETBP1-mutant disease.

cancer biology↗

bioRxiv2020

ASXL1 Directs Neutrophilic Differentiation via Modulation of MYC and RNA Polymerase II.

Mutations in the gene Additional Sex-Combs Like 1 (ASXL1) are recurrent in myeloid malignancies as well as the pre-malignant condition clonal hematopoiesis, where they are universally associated with poor prognosis. An epigenetic regulator, ASXL1 canonically directs the deposition of H3K27me3 via the polycomb repressive complex 2. However, its precise role in myeloid lineage maturation is incompletely described. We utilized single cell RNA sequencing (scRNA-seq) on a murine model of hematopoietic-specific ASXL1 deletion and identified a specific role for ASXL1 in terminal granulocyte maturation. Terminal maturation is accompanied by down regulation of Myc expression and cell cycle exit. ASXL1 deletion leads to hyperactivation of Myc in granulocyte precursors and a quantitative decrease in neutrophil production. This failure of normal developmentallyassociated Myc suppression is not accompanied by significant changes in the landscape of covalent histone modifications including H3K27me3. Examining the genome-wide localization of ASXL1 in myeloid progenitors revealed strong co-localization with RNA Polymerase II (RNAPII) at the promoters and spread across the gene bodies of transcriptionally active genes. ASXL1 deletion results in a decrease in RNAPII promoter-proximal pausing in granulocyte progenitors, indicative of a global increase in productive transcription, consistent with the known role of ASXL1 as a mediator of RNAPII pause release. These results suggest that ASXL1 inhibits productive transcription in granulocyte progenitors, identifying a new role for this epigenetic regulator and highlighting a novel potential oncogenic mechanism for ASXL1 mutations in myeloid malignancies.

cancer biology↗

bioRxiv2020

CBP/p300 orthologs CBP2 and CBP3 play distinct roles in planarian stem cell function

Chromatin modifications function as critical regulators of gene expression and cellular identity, especially in the regulation and maintenance of the pluripotent state. However, many studies of chromatin modification in stem cells--and pluripotent stem cells in particular--are performed in mammalian stem cell culture, an in vitro condition mimicking a very transient state during mammalian development. Thus, new models for study of pluripotent stem cells in vivo could be helpful for understanding the roles of chromatin modification, for confirming prior in vitro studies, and for exploring evolution of the pluripotent state. The freshwater flatworm, Schmidtea mediterranea, is an excellent model for studying adult pluripotent stem cells, particularly in the context of robust, whole-body regeneration. To identify chromatin modifying and remodeling enzymes critical for planarian regeneration and stem cell maintenance, we took a candidate approach and screened planarian homologs of 26 genes known to regulate chromatin biology in other organisms. Through our study, we identified six genes with novel functions in planarian homeostasis, regeneration, and behavior. We also identified in our list five planarian homologs of the mammalian CREB-Binding Protein (CBP) family of histone acetyltransferases, representing an expansion of this family in planarians. We find that two planarian CBP family members are required for planarian survival, with knockdown of Smed-CBP2 and Smed-CBP3 causing distinct defects in stem cell maintenance or function. Loss of CBP2 causes a quick, dramatic loss of stem cells, while knockdown of CBP3 more narrowly affects stem cells, preferentially decreasing markers of neural progenitors. We propose that the division of labor among a diversified CBP family in planarians presents an opportunity to dissect specific functions of a broadly important histone acetyltransferase family in stem cell biology.

developmental biology↗

bioRxiv2020

Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

The CRISPR/Cas12a RNA-guided complexes have a tremendous potential for nucleic acid detection due to its ability to indiscriminately cleave ssDNA once bound to a target DNA. However, the current CRISPR/Cas12a systems are limited to detecting DNA in a picomolar detection limit without an amplification step. Here, we developed a platform with engineered crRNAs and optimized conditions that enabled us to detect DNA, DNA/RNA heteroduplex and methylated DNA with higher sensitivity, achieving a limit of detection of in femtomolar range without any target pre-amplification step. By extending the 3- or 5-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discovered a new self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA. We applied this sensitive system to detect as low as 25 fM dsDNA from the PCA3 gene, an overexpressed biomarker in prostate cancer patients, in simulated urine over 6 hours. The same platform was used to detect as low as ~700 fM cDNA from HIV, 290 fM RNA from HCV, and 370 fM cDNA from SARS-CoV-2, all within 30 minutes without a need for target amplification. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs were incorporated in a paper-based lateral flow assay that could detect the target with up to 23-fold higher sensitivity within 40-60 minutes.

bioengineering↗

bioRxiv2019

Stereological characterization of the major cell lineages in the mouse dorsal dentate gyrus

Adult neurogenesis in the dorsal dentate gyrus (DG) subregion of the mammalian hippocampus supports critical cognitive processes related to memory. Local DG cell populations form a neurogenic niche specialized to regulate adult neurogenesis. Recently, DG astrocytes, microglia, endothelia, and neural stem cells have been identified as sources of neurogenesismodulating secreted factors. Accurately estimating the size of these cell populations is useful for elucidating their relative contributions to niche physiology. Previous studies have characterized these cell types individually, but to our knowledge no comprehensive study of all these cell types exists. This is problematic because considerable variability in reported population size complicates comparisons across studies. Here, we apply consistent stereological methods within a single study to estimate cell density for neurogenesis-modulating secretory cell types in the dorsal DG of adult mice. We used immunohistochemical phenotypic markers to quantify cell density and found that stellate astrocytes were the most numerous followed by endothelia, intermediate progenitors, microglia, and neural stem cells. We did not observe any significant sex differences in cell density. We expect our data will facilitate efforts to elucidate the role of DG secretory cell populations in regulating adult neurogenesis.

neuroscience↗