APOE3, but Not APOE4, Bone Marrow Transplantation Mitigates Behavioral and Pathological Changes in a Mouse Model of Alzheimer Disease

Apolipoprotein E4 (APOE4) genotype is the strongest genetic risk factor for late-onset Alzheimer disease and confers a proinflammatory, neurotoxic phenotype to microglia. Here, we tested the hypothesis that bone marrow cell APOE genotype modulates pathological progression in experimental Alzheimer disease. We performed bone marrow transplants (BMT) from green fluorescent protein–expressing human APOE3/3 or APOE4/4 donor mice into lethally irradiated 5-month-old APPswe/PS1ΔE9 mice. Eight months later, APOE4/4 BMT–recipient APPswe/PS1ΔE9 mice had significantly impaired spatial working memory and increased detergent-soluble and plaque Aβ compared with APOE3/3 BMT–recipient APPswe/PS1ΔE9 mice. BMT-derived microglia engraftment was significantly reduced in APOE4/4 recipients, who also had correspondingly less cerebral apoE. Gene expression analysis in cerebral cortex of APOE3/3 BMT recipients showed reduced expression of tumor necrosis factor-α and macrophage migration inhibitory factor (both neurotoxic cytokines) and elevated immunomodulatory IL-10 expression in APOE3/3 recipients compared with those that received APOE4/4 bone marrow. This was not due to detectable APOE-specific differences in expression of microglial major histocompatibility complex class II, C-C chemokine receptor (CCR) type 1, CCR2, CX3C chemokine receptor 1 (CX3CR1), or C5a anaphylatoxin chemotactic receptor (C5aR). Together, these findings suggest that BMT-derived APOE3-expressing cells are superior to those that express APOE4 in their ability to mitigate the behavioral and neuropathological changes in experimental Alzheimer disease

VERITAS discovery of very high energy gamma-ray emission from S3 1227+25 and multiwavelength observations

We report the detection of very high energy gamma-ray emission from the blazar S3 1227+25 (VER J1230+253) with the Very Energetic Radiation Imaging Telescope Array System (VERITAS). VERITAS observations of the source were triggered by the detection of a hard-spectrum GeV flare on May 15, 2015 with the Fermi-Large Area Telescope (LAT). A combined five-hour VERITAS exposure on May 16th and May 18th resulted in a strong 13$\sigma$ detection with a differential photon spectral index, $\Gamma$ = 3.8 $\pm$ 0.4, and a flux level at 9% of the Crab Nebula above 120 GeV. This also triggered target of opportunity observations with Swift, optical photometry, polarimetry and radio measurements, also presented in this work, in addition to the VERITAS and Fermi-LAT data. A temporal analysis of the gamma-ray flux during this period finds evidence of a shortest variability timescale of $\tau_{obs}$ = 6.2 $\pm$ 0.9 hours, indicating emission from compact regions within the jet, and the combined gamma-ray spectrum shows no strong evidence of a spectral cut-off. An investigation into correlations between the multiwavelength observations found evidence of optical and gamma-ray correlations, suggesting a single-zone model of emission. Finally, the multiwavelength spectral energy distribution is well described by a simple one-zone leptonic synchrotron self-Compton radiation model.Comment: 18 pages, 6 figures. Accepted for publication in the Astrophysical Journal (ApJ

Comparative cellular analysis of motor cortex in human, marmoset and mouse

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties

Regeneration of retinal neurons from Müller glia in adult mice

Thesis (Ph.D.)--University of Washington, 2019There are many diseases that result in the death of specific neuron populations in the retina, resulting in blindness. Currently, few options exist for the treatment of blinding diseases, and the options that are available can be invasive and only have modest efficacy. Various non-mammalian organisms have the natural ability to regenerate their retinal neurons following injury. The proneural transcription factor, Ascl1, has been shown to be necessary and sufficient to initiate the regenerative process in these species, specifically in the retinal Müller glia. After Ascl1 induction, the Müller glia proliferate, migrate, and differentiate into all subtypes of retinal neurons and even integrate into the retinal circuitry, fully restoring function to the animal. I have identified a method for stimulating the regeneration of specific neuron subtypes in mammals by overexpressing Ascl1 in the Müller glia of adult mice. These Müller glial-derived neurons resemble nascent retinal neurons at the morphological, protein, RNA, and epigenetic levels. Additionally, the newly generated neurons integrate into the existing retinal circuitry and response to light stimuli similar to nascent neurons. In this dissertation, I will discuss how we achieved retinal regeneration in adult mice and studies that were performed to further improve and characterize the Müller glia-derived neurons

Suppressed retinal degeneration in aged wild type and APPswe/PS1ΔE9 mice by bone marrow transplantation.

Alzheimer's disease (AD) is an age-related condition characterized by accumulation of neurotoxic amyloid β peptides (Aβ) in brain and retina. Because bone marrow transplantation (BMT) results in decreased cerebral Aβ in experimental AD, we hypothesized that BMT would mitigate retinal neurotoxicity through decreased retinal Aβ. To test this, we performed BMT in APPswe/PS1ΔE9 double transgenic mice using green fluorescent protein expressing wild type (wt) mice as marrow donors. We first examined retinas from control, non-transplanted, aged AD mice and found a two-fold increase in microglia compared with wt mice, prominent inner retinal Aβ and paired helical filament-tau, and decreased retinal ganglion cell layer neurons. BMT resulted in near complete replacement of host retinal microglia with BMT-derived cells and normalized total AD retinal microglia to non-transplanted wt levels. Aβ and paired helical filament-tau were reduced (61.0% and 44.1% respectively) in BMT-recipient AD mice, which had 20.8% more retinal ganglion cell layer neurons than non-transplanted AD controls. Interestingly, aged wt BMT recipients also had significantly more neurons (25.4%) compared with non-transplanted aged wt controls. Quantitation of retinal ganglion cell layer neurons in young mice confirmed age-related retinal degeneration was mitigated by BMT. We found increased MHC class II expression in BMT-derived microglia and decreased oxidative damage in retinal ganglion cell layer neurons. Thus, BMT is neuroprotective in age-related as well as AD-related retinal degeneration, and may be a result of alterations in innate immune function and oxidative stress in BMT recipient mice

Increased microglia density in experimental AD is mitigated by BMT.

<p><b>A</b>: Representative retinal cryosections from wt (left) and APP<i>swe</i>-PS1ΔE9 mice (right) were stained with anti-Iba-1 antibody and visualized with Cy3-conjugated secondary antibody. Ramified microglia were primarily identified in ganglion cell layer (gcl), inner plexiform layer (ipl) and outer plexiform layer (opl) in wt and APP<i>swe</i>-PS1ΔE9 retinas. <b>B</b>: Unbiased stereologic analysis revealed increased microglia density in control (untreated) APP<i>swe</i>-PS1ΔE9 retina compared with wt control mice (*<i>P</i><0.05, n = 7–9, student's <i>t</i> test). <b>C</b>: Average Iba-1⁺ microglia density in the different retinal layers. Significantly higher numbers of Iba-1⁺ microglia were noted in gcl, ipl and opl in APP<i>swe</i>-PS1ΔE9 mice (*<i>P</i><0.05, ***<i>P</i><0.001, n = 7–9, two-way ANOVA followed by Bonferroni <i>post</i> test.) Confocal images from wt (<b>D</b>) and APP<i>swe</i>-PS1ΔE9 (<b>E</b>) retinas immunostained with Iba-1 (red) demonstrate that BM-derived GFP⁺ cells (green) exhibited ramified microglia morphology and had near-uniform expression of Iba-1. In both wt and APP<i>swe</i>-PS1ΔE9 BMT recipient mice, host microglia were almost completely replaced with BM-derived cells. <b>F</b>: Unbiased stereologic quantitation of retinal microglia engraftment revealed 91.2±4.0% of wt and 73.2±16.0% of APP<i>swe</i>-PS1ΔE9 Iba-1⁺ microglia were BM derived (GFP⁺). <b>G</b>: Microglia density in retina of wt BMT recipients was not different from non-transplanted wt mice, but BMT in APP<i>swe</i>-PS1ΔE9 mice normalized microglia density to wt levels. Data are percent of wt Iba-1⁺ cells/mm² in non-transplanted APP<i>swe</i>-PS1ΔE9 untreated mice (AD no Tx) or APP<i>swe</i>-PS1ΔE9 and wt BMT recipients (*<i>P</i><0.05, **<i>P</i><0.01, n = 6–10, one-way ANOVA followed by Bonferroni <i>post</i> test). Scale bars  = 30 μm.</p