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

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

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

Aβ and PHF-tau are reduced in retina of APP<i>swe</i>-PS1ΔE9 BMT recipient mice.

<p><b>A</b>: Representative photomicrographs of Aβ deposition in non-transplanted, age-matched APP<i>swe</i>-PS1ΔE9 control retina (top, AD No Tx) or APP<i>swe</i>-PS1ΔE9 that received BMT (bottom, AD BMT) stained with anti-Aβ antibody and visualized with Cy3-conjugated secondary antibody (red). Region of inset is indicated by arrows. Scale bar  = 50 µm. <b>B</b>: Quantitative analysis of Aβ immunofluorescence using a standardized digital thresholding protocol demonstrated significant reduction in retinal Aβ in BMT APP<i>swe</i>-PS1ΔE9 mice compared with non-transplanted APP<i>swe</i>-PS1ΔE9 control mice (***<i>P</i><0.001, n = 6, student's <i>t</i> test). <b>C</b>: Representative photomicrographs of PHF-tau immunofluorescence in retinal ganglion cell layer (RGCL) (arrows) of non-transplanted, age-matched control APP<i>swe</i>-PS1ΔE9 mice (top, AD No Tx) compared with APP<i>swe</i>-PS1ΔE9 BMT recipients (bottom, AD BMT). Nuclei were counterstained with DAPI (blue). Scale bar  = 30 µm. <b>D</b>: Quantitative analysis of PHF-tau immunofluorescence using a standardized digital thresholding protocol demonstrated significant reduction of PHF-tau in APP<i>swe</i>-PS1ΔE9 BMT-recipients compared with non-transplanted controls (*<i>P</i><0.05, n = 6, one-way ANOVA analysis with Bonferroni <i>post</i> test).</p

BMT mediates neuroprotection of RGCL neurons in APP<i>swe</i>-PS1ΔE9 and wt mice.

<p><b>A–C</b>: RGCL neurons were identified using anti-NeuN antibody and visualized with Cy3-conjugated secondary antibody in 13-month-old APP<i>swe</i>-PS1ΔE9 and wt mice. Representative photomicrographs of NeuN⁺ RGCL neurons in control (A, top) and BMT-recipient (A, bottom) APP<i>swe</i>-PS1ΔE9 mice demonstrate neuroprotective effects of BMT through preservation of RGCL neurons (B) and inner retinal (NFL+RGCL+IPL) thickness (C). <b>D–E</b>: Representative photomicrographs of NeuN⁺ RGCL neurons in 13-month-old control (D, top) and BMT-recipient (D, bottom) wt mice also demonstrate neuroprotective effects of BMT through preservation of RGCL neurons (E). <b>F</b>: No effects on retinal thickness in wt recipients compared with the wt controls. *<i>P</i><0.05, n = 6–10, student's <i>t</i> test. Scale bar  = 50 μm.</p

RGCL neuroprotection is not due to effects of high dose cranial irradiation alone.

<p><b>A</b>: Representative photographs of control mice (no Tx) and mice that received head only irradiation (HO-XRT) demonstrate effects of irradiation on coat color and confirm radiation exposure in HO-XRT mice (top). Representative photomicrographs of NeuN⁺ neurons (red) in RGCL stained with NeuN antibody and visualized with Cy3-conjugated secondary antibody show a mild reduction in neuron density 8 months after HO-XRT (bottom). Scale bar  = 50 μm. <b>B</b>: Quantification of neuron density depicted as a percent of age-matched, non-irradiated wt controls demonstrates there is a mild reduction in RGCL neurons as a result of high dose irradiation without BMT in wt and APP<i>swe</i>-PS1ΔE9 mice, thus eliminating the possibility that high dose cranial irradiation underlies the neuroprotective effects of BMT.</p