Biology subjects

Freeman, W. M.

Publications and source records attributed to Freeman, W. M..

25 records · Page 2Linked to original sources

bioRxiv2021

Minimizing the ex vivo confounds of cell-isolation techniques on transcriptomic profiles of purified microglia

Modern molecular neuroscience studies require analysis of specific cellular populations derived from brain tissue samples to disambiguate cell-type specific events. This is particularly true in the analysis of minority glial populations in the brain, such as microglia, which may be obscured in whole tissue analyses. Microglia have central functions in development, aging, and neurodegeneration and are a current focus of neuroscience research. A long-standing concern for glial biologists using in vivo models is whether cell isolation from CNS tissue could introduce ex vivo artifacts in microglia, which respond quickly to changes in the environment. Mouse microglia were purified by magnetic-activated cell sorting (MACS), as well as cytometer- and cartridge-based fluorescence-activated cell sorting (FACS) approaches to compare and contrast performance. The Cx3cr1-NuTRAP mouse model was used here to provide an endogenous fluorescent microglial marker and a microglial-specific translatome profile as a baseline comparison lacking cell isolation artifacts. All methods performed similarly for microglial purity with main differences being in cell yield and time of isolation. Ex vivo activation signatures occurred principally during the initial tissue dissociation and cell preparation and not the microglial cell sorting. Utilizing transcriptional and translational inhibitors during the cell preparation prevented the activational phenotype. These data demonstrate that a variety of microglial isolation approaches can be used, depending on experimental needs, and that inhibitor cocktails are effective at reducing cell preparation artifacts. Table of Contents Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/452509v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@823562org.highwire.dtl.DTLVardef@7edb25org.highwire.dtl.DTLVardef@656feeorg.highwire.dtl.DTLVardef@197aae1_HPS_FORMAT_FIGEXP M_FIG C_FIG Main PointsMACS, cytometer-FACS, and cartridge-FACS give equivalent and sufficient yield/purity for microglial analyses. Ex vivo microglial activation is prevented by supplementation with transcription/translation inhibitors during cell preparation.

neuroscience↗

bioRxiv2021

The NOTCH3 Downstream Target HEYL Regulates Human Airway Epithelial Club Cell Differentiation

Basal cells (BC) are the resident stem/progenitor cells of the adult pseudostratified airway epithelium, whose differentiation program is orchestrated by the NOTCH signaling pathway. NOTCH3 receptor mediated signaling regulates BC to club cell differentiation; however, the downstream responses that regulate this process are largely unknown. In the present study we used an in vitro air-liquid interface model of the human pseudostratified airway epithelium to identify the NOTCH3-dependent downstream genes/pathways that regulate human BC to club cell differentiation. Activation of NOTCH3 signaling in BC via lentivirus-mediated over-expression of the active NOTCH3 intracellular domain (NICD3) promoted club cell differentiation. Bulk RNA-seq analysis of control vs NICD3-transduced cells, identified 692 NICD3 responsive genes enriched for pathways linked to airway epithelial biology and differentiation including Wnt/{beta}-catenin Signaling. Expression of the classical NOTCH target HEYL increased in response to NOTCH3 activation and positively correlated with expression of the club cell marker SCGB1A1. Further, using single-cell RNA-seq, we report that HEYL+ cells primarily clustered with SCGB1A1+ and NOTCH3+ cells. Moreover, HEYL protein co-localized with SCGB1A1 in ALI cultures in vitro and in the human and mouse airway epithelium in vivo. siRNA-mediated knockdown of HEYL in BC led to changes in epithelial structure including altered morphology and significant reductions in transepithelial electrical resistance and expression of tight junction related genes. Finally, HEYL knockdown significantly reduced the number of SCGB1A1+ club cells, along with a corresponding increase in KRT8+ BC-intermediate cells. Overall, our data identifies NOTCH3-HEYL signaling as a key regulator of BC to club cell differentiation.

cell biology↗

bioRxiv2020

Litter expansion alters metabolic homeostasis in a sexually divergent manner

Nutritional manipulations early in life have been shown to influence growth rate and elicit long lasting effects which in turn has been found to impact lifespan. Therefore, we studied the long-term effects of pre-weaning dietary restriction implemented by litter expansion (4, 6, 8, 10, and 12 pups per dam: LS4, LS6, LS8, LS10, LS12) on male and female C57BL/6 mice. After weaning, these mice were fed ad libitum a commercial lab chow for the 15-month duration of the study. The mice from large litter sizes (LS12) were significantly leaner and had reduced total fat mass compared to the normal size litters (LS 6) starting from weaning through to 15 months of age. Male LS10 & 12 mice showed significant reduction in their fat depot masses at 15 months of age: gonadal, subcutaneous, and brown fat whereas the females did not mimic these findings. At 9 months of age, both male and female LS10 and 12 mice showed improved glucose tolerance; however, only male LS10 and LS12 mice showed improved insulin tolerance starting at 5 months of age. In addition, we found that the male LS8, 10 & 12 mice at 15 months of age showed significantly reduced IGF-1 levels in the serum and various other organs (liver, gastrocnemius and brain cortex). Interestingly, the female LS8, 10, 12 mice showed a different pattern with reduced IGF-1 levels in serum, liver and gastrocnemius but not in the brain cortex. Similarly, the litter expanded mice showed sexual divergence in levels of FGF21 and adiponectin with only the male mice showing increased FGF21 and adiponectin levels at 15 months of age. In summary, our data show that, litter expansion results in long-lasting metabolic changes that are age and sex dependent with the male mice showing an early and robust response compared to female mice.

molecular biology↗

bioRxiv2020

Health benefits attributed to 17α-estradiol, a lifespan-extending compound, are mediated through estrogen receptor α

Metabolic dysfunction underlies several chronic diseases, many of which are exacerbated by obesity. Dietary interventions can reverse metabolic declines and slow aging, although compliance issues remain paramount. 17-estradiol treatment improves metabolic parameters and slows aging in male mice. The mechanisms by which 17-estradiol elicits these benefits remain unresolved. Herein, we show that 17-estradiol elicits similar genomic binding and transcriptional activation through estrogen receptor (ER) to that of 17{beta}-estradiol. In addition, we show that the ablation of ER completely attenuates the beneficial metabolic effects of 17-E2 in male mice. Our findings suggest that 17-E2 acts primarily through the liver and hypothalamus to improve metabolic parameters in male mice. Lastly, we also determined that 17-E2 improves metabolic parameters in male rats, thereby proving that the beneficial effects of 17-E2 are not limited to mice. Collectively, these studies suggest ER may be a drug target for mitigating chronic diseases in male mammals.

physiology↗

bioRxiv2019

Inducible cell-specific mouse models for paired epigenetic and transcriptomic studies of microglia and astroglia

Epigenetic regulation of gene expression occurs in a cell type-specific manner. Current cell-type specific neuroepigenetic studies rely on cell sorting methods that can alter cell phenotype and introduce potential confounds. Here we demonstrate and validate a Nuclear Tagging and Translating Ribosome Affinity Purification (NuTRAP) approach for temporally controlled labeling and isolation of ribosomes and nuclei, and thus RNA and DNA, from specific CNS cell types. Paired analysis of the transcriptome and DNA modifications in astrocytes and microglia demonstrates differential usage of DNA methylation and hydroxymethylation in CG and non-CG contexts that corresponds to cell type-specific gene expression. Application of this approach in LPS treated mice uncovers microglia-specific transcriptome and epigenome changes in inflammatory pathways that cannot be detected with tissue-level analysis. The NuTRAP model and the validation approaches presented can be applied to any CNS cell type for which a cell type-specific cre is available.

neuroscience↗

bioRxiv2019

Female mice are resilient to age-related decline of substantia nigra dopamine neuron firing parameters

The degeneration of substantia nigra (SN) dopamine neurons is a central feature in the pathology associated with Parkinsons disease, which is characterized by progressive loss of motor and cognitive functions. The largest risk factors for Parkinsons disease are age and sex; most cases occur after age 60 and males have nearly twice the incidence as females. While much research in Parkinsons has focused on the mechanisms underlying dopamine neuron degeneration, very little work has considered the influence of these two risk factors to disease risk and presentation. In this work, we performed whole cell patch clamp recordings in brain slices to study the alterations in intrinsic firing properties of single dopamine neurons in C57BL/6 mice across ages and between sexes. We observed a progressive decline in dopamine neuron firing activity in males by 18 months of age, while dopamine neurons from females remained largely unaffected. A semiquantitative analysis of midbrain dopamine neuron populations revealed a slight decrease only in substantia nigra dopamine neurons in males, while females did not change. This was also accompanied by increases in the expression of genes that have been linked to Parkinsons including PTEN-induced kinase 1 (PINK1) in both males and females, and the ubiquitin ligase parkin, primarily in the substantia nigra of males. These impairments in dopamine neuron function in males may represent a vulnerability to further insults that could predispose these cells to neurodegenerative diseases such as in Parkinsons.

neuroscience↗

bioRxiv2019

Early life DNA methylation profiles are indicative of age-related transcriptome changes

Alterations to cellular and molecular programs with brain aging result in cognitive impairment and susceptibility to neurodegenerative disease. Changes in DNA methylation patterns, an epigenetic modification required for various CNS functions, are observed with aging and can be prevented by anti-aging interventions, but the functional outcomes of altered methylation on transcriptome profiles are poorly understood with brain aging. Integrated analysis of the hippocampal methylome and transcriptome with aging of male and female mice demonstrates that age-related differences in methylation and gene expression are anti-correlated within gene bodies and enhancers, but not promoters. Methylation levels at young age of genes altered with aging are positively associated with age-related expression changes even in the absence of significant changes to methylation with aging, a finding also observed in mouse Alzheimers models. DNA methylation patterns established in youth, in combination with other epigenetic marks, are able to predict changes in transcript trajectories with aging. These findings are consistent with the developmental origins of disease hypothesis and indicate that epigenetic variability in early life may explain differences in age-related disease.

genomics↗