Biology subjects

Hildebrand, S.

Publications and source records attributed to Hildebrand, S..

26 records · Page 2Linked to original sources

bioRxiv2022

Silencing of ApoE with Divalent siRNAs Drives Activation of Immune Clearance Pathways and Improves Amyloid Pathology in Mouse Models of Alzheimer's Disease

The most common genetic risk factor for late-onset Alzheimers disease (AD) is the APOE4 allele, with evidence for gain- and loss-of-function mechanisms. ApoE knockout in mice abrogates AD phenotypes but causes severe atherosclerosis due to the role of liver ApoE in cholesterol homeostasis. Previous attempts to inhibit brain-specific ApoE with anti-sense oligonucleotides only modestly reduced ApoE expression and had no effect on amyloid burden in adult AD mice. Here, we optimized a divalent small interfering RNA (di-siRNA) to selectively and potently silence ApoE in the brain. Silencing brain ApoE in AD mice significantly reduced amyloid plaque formation without affecting systemic cholesterol levels, confirming that brain and liver APOE pools are spatially and functionally distinct. Mechanistically, APOE appears to be a scaffold for beta-amyloid aggregation that limits clearance by microglia. Di-siRNAs from this study can be taken to pre-clinical and clinical trials to accelerate development of AD-modifying therapies and establish siRNA-based modulation of ApoE as a viable path towards therapeutic development.

neuroscience↗

bioRxiv2021

De novo germline mutation in the Dual Specificity Phosphatase 10 gene accelerates autoimmune diabetes in Non-Obese Diabetic (NOD) mice

Here we report the isolation by selective breeding of two sublines of Non-Obese Diabetic (NOD) mice exhibiting a significant difference in the incidence of autoimmune type 1 diabetes (T1D). Whole genome sequencing of the NOD/NckH (high T1D incidence) and NOD/NckL (low T1D incidence) revealed the presence of a limited number of variants specific to each subline. Treating the age of T1D onset as a quantitative trait and using automated meiotic mapping (AMM), enhanced susceptibility in the NOD/NckH subline was unambiguously attributed to a recessive allele of Dusp10 which encodes a dual specificity phosphatase. The causative effect of the mutation was verified with a high level of confidence by targeting Dusp10 with CRISPR/Cas9 in NOD/NckL mice: in these animals a higher incidence of diabetes was observed. Expression of wild-type Dusp10 correlated with higher levels of surface PD-L1 in the islets of NOD/NckL mice.

immunology↗

bioRxiv2020

Candidate Explorer: a tool for discovery, evaluation, and display of mutations causing significant immune phenotypes

When applied to immunity, forward genetic studies use meiotic mapping to provide strong statistical evidence that a particular mutation is causative of a particular immune phenotype. Notwithstanding this, co-segregation of multiple mutations, occasional unawareness of mutations, and paucity of homozygotes may lead to erroneous declarations of cause and effect. We sought to improve the selection of authentic causative mutations using a machine learning software tool, Candidate Explorer (CE), which integrates 65 data features into a single numeric score, mathematically convertible to the likelihood of verification of any putative mutation-phenotype association. CE has identified most genes within which mutations can be causative of flow cytometric phenovariation in Mus musculus. The majority of these genes were not previously known to support immune function or homeostasis. Mouse geneticists will find CE data informative in identifying causative mutations within quantitative trait loci, while clinical geneticists may use CE to help connect causative variants with rare heritable diseases of immunity, even in the absence of linkage information. CE displays integrated mutation, phenotype, and linkage data, and is freely available for query online.

genetics↗

bioRxiv2020

Mechanism of negative regulation of NF-κB by N4BP1

Many immune responses depend upon activation of NF-{kappa}B, a key transcription factor in the elicitation of a cytokine response. Here we demonstrate that N4BP1 inhibited TLR-dependent activation of NF-{kappa}B by interacting with the NF-{kappa}B signaling essential modulator (NEMO, also known as I{kappa}B kinase {gamma}) to attenuate NEMO-NEMO dimerization or oligomerization. The UBA-like (ubiquitin associated-like) and CUE-like (ubiquitin conjugation to ER degradation) domains in N4BP1 mediated the interaction with the NEMO COZI domain. Both in vitro and in mice, N4bp1 deficiency specifically enhanced TRIF-independent (TLR2, TLR7, or TLR9-mediated), but not TRIF-dependent (TLR3 or TLR4-mediated), NF-{kappa}B activation leading to increased production of proinflammatory cytokines. In response to TLR4 or TLR3 activation, TRIF caused activation of caspase 8, which cleaved N4BP1 distal to residues D424 and D490 and abolished its inhibitory effect. N4bp1-/- mice also exhibited diminished numbers of T cells in the peripheral blood. Our work identifies N4BP1 as an inhibitory checkpoint protein that must be overcome to activate NF-{kappa}B, and a TRIF-initiated caspase 8-dependent mechanism by which this is accomplished.

immunology↗

bioRxiv2020

Highly efficient genome modification of cultured primordial germ cells with lentiviral vectors to generate transgenic songbirds

SUMARYThe ability to genetically manipulate organisms has led to significant insights in functional genomics in many species. In birds, manipulation of the genome is hindered by the inaccessibility of the one-cell embryo. During embryonic development, avian primordial germ cells (PGCs) migrate through the blood stream and reach the gonadal anlage; where they develop into mature germ cells. Here, we explored the use of PGCs to produce transgenic offspring in the zebra finch, which is a major animal model for sexual brain differentiation, vocal learning and vocal communication. Zebra finch PGCs (zfPGCs) obtained from embryonic blood significantly proliferated when cultured in an optimized culture medium and conserved the expression of germ and stem cell markers. Transduction of cultured zfPGCs with lentiviral vectors was highly efficient leading to strong expression of the enhanced green fluorescent protein (eGFP). Transduced zfPGCs were injected into the host embryo and transgenic songbirds were successfully generated.

neuroscience↗

bioRxiv2019

Monte Carlo Simulation of Diffusion MRI in geometries constructed from two-photon microscopy of cortical gray matter

PurposeNeurodegenerative diseases such as Alzheimers disease cause changes and disruption to cortical microstructure and architecture. Diffusion MRI (dMRI) could potentially be sensitive to such changes. There is a growing interest in modeling of human cortical areas using a combination of quantitative MRI and 3D microscopy. The purpose of this study was to quantitatively characterize the cytoarchitecture of human cortical tissue from 3D fluorescence microscopy to simulate diffusion MRI (dMRI) signal in the cortex to better understand its diffusion signal characteristics.\n\nMethodsDiffusion of water molecules and dMRI signal were simulated by an indirect geometry based method and a direct voxel based method in microstructural details extracted from microscopy of cortex. Additionally, residence times of diffusing spins inside voxel volumes were considered to set effective resolution limits. Mean diffusivity (MD) and kurtosis (MK) were calculated for variable cell and neurite densities, sizes and diffusion times under realistic values for permeability and free diffusion.\n\nResultsBoth simulation methods could efficiently and accurately simulate dMRI signals with fractional anisotropy, diffusion coefficient and kurtosis in agreement with previous reports. Simulated MD and MK showed changes with increasing diffusion times specific to cortical cell density and sizes, with MK showing the highest sensitivity. Intra-voxel residence times with increasing diffusion times showed that the effective dMRI resolution approaches the thickness of cortical layers.\n\nConclusionsMonte Carlo simulations based on 3D microscopy data enable estimating changes in MD and MK over diffusion times and are sensitive to cortical cytoarchitecture and its possible changes in neurodegenerative disease. When considering layer-specific cortical dMRI, effective resolution due to residence times is an important concern.

biophysics↗

bioRxiv2019

The mesoSPIM initiative: open-source light-sheet mesoscopes for imaging in cleared tissue

Over the course of the past decade, tissue clearing methods have reached a high level of sophistication with a wide variety of approaches now available1. To image large cleared samples, light-sheet microscopes have proven to be ideal due to their excellent optical sectioning capability in transparent tissue2. Such instruments have recently seen extensive technological and commercial development. However, despite this progress, the community is lacking instruments capable of exploring large samples with near-isotropic resolution within minutes. Here, we introduce the mesoscale selective plane-illumination microscopy (mesoSPIM) initiative, an open-hardware project that provides researchers with instructions and software to easily build and operate light-sheet microscopes for centimeter-sized cleared samples (http://www.mesospim.org).\n\nA wide range of commercial light-sheet microscopes have ...

neuroscience↗

bioRxiv2018

Scalable cytoarchitectonic characterization of large intact human neocortex samples

We describe MASH (Multiscale Architectonic Staining of Human cortex): a simple, fast and low-cost cytoarchitectonic labeling and optical clearing approach for human cortex samples, which can be applied to large formalin fixed adult brain samples. A suite of small-molecule fluorescent nuclear and cytoplasmic dyes in combination with new refractive index matching solutions allows deep volume imaging. This enables highly scalable human neocortical cytoarchitecture characterization with a large 3D scope.

neuroscience↗