Biology subjects

Nitsch, R. M.

Publications and source records attributed to Nitsch, R. M..

24 records · Page 2Linked to original sources

bioRxiv2021

APOE2, E3 and E4 differentially modulate cellular homeostasis, cholesterol metabolism and inflammatory response in isogenic iPSC-derived astrocytes

Apolipoprotein E (APOE) is the principal lipid carrier in the CNS and mainly expressed by astrocytes. The three different APOE alleles (E2, E3, and E4) impose differential risk to Alzheimers disease (AD); E2 is protective, E3 is defined as average risk, while E4 is the major genetic risk factor for sporadic AD. Despite recent advances, the fundamental role of different APOE alleles in brain homeostasis is still poorly understood. To uncover the functional role of APOE in human astrocytes, we differentiated human APOE-isogenic iPSCs (E4, E3, E2 and APOE-knockout (KO)) to functional astrocytes (hereafter "iAstrocytes"), with a resting, non-proliferating phenotype. Functional assays indicated that polymorphisms in APOE (APOE4>E3>E2=KO) reduced iAstrocyte metabolic and clearance functions including glutamate uptake and receptor-mediated uptake of {beta}-amyloid aggregates. We performed unlabelled mass spectrometry-based proteomic analysis of iAstrocytes at baseline and after activation with interleukin-1{beta} (IL-1{beta}) showing a reduction of cholesterol and lipid metabolic and biosynthetic pathways, and an increase of immunoregulatory pathways at baseline (E4>E3>E2). Cholesterol efflux and biosynthesis were reduced in E4 iAstrocytes, and subcellular localization of cholesterol in lysosomes was increased. In APOE-KO iAstrocytes, APOE-independent mechanisms showed to be proficient in mediating cholesterol biosynthesis and efflux. Proteomic analysis of IL-1{beta}-treated iAstrocytes showed an increase of cholesterol/lipid metabolism and biosynthesis as well as inflammatory pathways. Furthermore, cholesterol efflux, which was reduced in APOE4 iAstrocytes at baseline, was alleviated in activated E4 iAstrocytes. Inflammatory cytokine release was exacerbated upon IL-1{beta} treatment in E4 iAstrocytes (E4>E3>E2>KO), in line with the proteomic data. Taken together, we show that APOE plays a major role in several physiological and metabolic processes in human astrocytes with APOE4 pushing iAstrocytes to a disease-relevant phenotype, causing dysregulated cholesterol/lipid homeostasis, increased inflammatory signalling and reduced {beta}-amyloid uptake while APOE2 iAstrocytes show opposing effects. Our study provides a new reference for AD-relevant proteomic and metabolic changes, mediated by the three main APOE isoforms in human astrocytes.

neuroscience↗

bioRxiv2021

Non-invasive imaging of tau-targeted probe uptake by whole brain multi-spectral optoacoustic tomography

AimAbnormal tau accumulation plays an important role in tauopathy diseases such as Alzheimers disease and Frontotemporal dementia. There is a need for high-resolution imaging of tau deposits at the whole brain scale in animal models. Here, we demonstrate non-invasive whole brain imaging of tau-targeted PBB5 probe in P301L model of 4-repeat tau at 130 m resolution using volumetric multi-spectral optoacoustic tomography (vMSOT). MethodsThe binding properties of PBB5 to 4-repeat K18 tau and A{beta}42 fibrils were assessed by using Thioflavin T assay and surface plasmon resonance assay. We identified the probe PBB5 suitable for vMSOT tau imaging. The imaging performance was first evaluated using postmortem human brain tissues from patients with Alzheimers disease, corticobasal degeneration and progressive supranuclear palsy. Concurrent vMSOT and epi-fluorescence imaging of in vivo PBB5 targeting (i.v.) was performed in P301L and wild-type mice. Ex vivo measurements on excised brains along with multiphoton microscopy and immunofluorescence staining of tissue sections were performed for validation. The spectrally-unmixed vMSOT data was registered with MRI atlas for volume-of-interest analysis. ResultsPBB5 showed specific binding to recombinant K18 tau fibrils, Alzheimers disease brain tissue homogenate by competitive binding against [11C]PBB3 and to tau deposits (AT-8 positive) in post-mortem corticobasal degeneration and progressive supranuclear palsy brain. i.v. administration of PBB5 in P301L mice led to retention of the probe in tau-laden cortex and hippocampus in contrast to wild-type animals, as also confirmed by ex vivo vMSOT, epi-fluorescence and multiphoton microscopy results. ConclusionvMSOT with PBB5 facilitates novel 3D whole brain imaging of tau in P301L animal model with high-resolution for future mechanistic studies and monitoring of putative treatments targeting tau.

neuroscience↗

bioRxiv2020

DTI reveals whole-brain microstructural changes in the P301L mouse model of tauopathy

IntroductionIncreased expression of hyperphosphorylated tau and the formation of neurofibrillary tangles are associated with neuronal loss and white matter damage. Using high resolution ex vivo diffusion tensor imaging (DTI), we investigated microstructural changes in the white and grey matter in the P301L mouse model of human tauopathy at 8.5 months-of-age. For unbiased computational analysis, we implemented a pipeline for voxel-based analysis (VBA) and atlas-based analysis (ABA) of DTI mouse brain data. MethodsHemizygous and homozygous transgenic P301L mice and non-transgenic littermates were used. DTI data were acquired for generation of fractional anisotropy (FA), mean diffusivity (MD), radial diffusivity (RD), axial diffusivity (AD) maps. VBA on the entire brain were performed using SPM8 and SPM Mouse toolbox. Initially, all DTI maps were co-registered with Allen mouse brain atlas to bring them to one common coordinate space. In VBA, co-registered DTI maps were normalized and smoothed in order to perform two-sample t-tests to compare hemizygotes with non-transgenic littermates, homozygotes with non-transgenic littermates, hemizygotes with homozygotes on each DTI parameter map. In ABA, the average values for selected regions-of-interests were computed with co-registered DTI maps and labels in Allen mouse brain atlas. After, the same two-sample t-tests were executed on the estimated average values. ResultsWe made reconstructed DTI data and VBA and ABA pipeline publicly available. With VBA, we found microstructural changes in the white matter in hemizygous P301L mice compared to non-transgenic littermates. In contrast, more pronounced and brain-wide spread changes were observed in VBA when comparing homozygous P301L mice with non-transgenic littermates. Statistical comparison of DTI metrics in selected brain regions by ABA corroborated findings from VBA. FA was found to be decreased in most brain regions, while MD, RD and AD were increased compared to hemizygotes and non-transgenic littermates. Discussion/ConclusionHigh resolution ex vivo DTI demonstrated brain-wide microstructural changes in the P301L mouse model of human tauopathy. The comparison between hemizygous and homozygous P301L mice revealed a gene-dose dependent effect on DTI metrics. The publicly available computational data analysis pipeline can provide a platform for future mechanistic and longitudinal studies.

neuroscience↗

bioRxiv2020

Whole brain optoacoustic tomography reveals strain-specific regional beta-amyloid densities in Alzheimer`s disease amyloidosis models

Deposition of beta-amyloid (A{beta}) deposits is one major histopathological hallmark of Alzheimers disease (AD). Here, we introduce volumetric multi-spectral optoacoustic tomography (vMSOT), which covers 10x10x10 mm3 field-of-view, capable of 3D whole mouse brain imaging. We show for the first time the optoacoustic properties of oxazine-derivative AOI987 probe, which binds to A{beta}, and the application of vMSOT for the quantification of brain-wide A{beta} deposition. Administration of AOI987 to two common transgenic mouse strains of AD amyloidosis led to a retention of the probe in A{beta}-laden brain regions. Co-registered of vMSOT data to a brain atlas revealed strain-specific pattern of AOI987 uptake. A comparison with ex vivo light-sheet microscopy in cleared mouse brains showed a good correspondence in A{beta} distribution. Lastly, we demonstrate the specificity of the AOI987 probe by immunohistochemistry. vMSOT with AOI987 facilitates preclinical brain region-specific studies of A{beta} spread and accumulation, and the monitoring of putative treatments targeting A{beta}.

neuroscience↗

bioRxiv2020

Transcranial in vivo detection of Aβ at single plaque resolution with large-field multifocal illumination fluorescence microscopy

The abnormal deposition of beta-amyloid proteins in the brain is one of the major histopathological hallmarks of Alzheimers disease. Currently available intravital microscopy techniques for high-resolution plaque visualization commonly involve highly invasive procedures and are limited to a small field-of-view within the rodent brain. Here, we report the transcranial detection of amyloid-beta deposits at the whole brain scale with 20 m resolution in APP/PS1 and arcA{beta} mouse models of Alzheimers disease amyloidosis using a large-field multifocal (LMI) fluorescence microscopy technique. Highly sensitive and specific detection of amyloid-beta deposits at a single plaque level in APP/PS1 and arcA{beta} mice was facilitated using luminescent conjugated oligothiophene HS-169. Immunohistochemical staining with HS-169, anti-A{beta} antibody 6E10, and conformation antibodies OC (fibrillar) of brain tissue sections further showed that HS-169 resolved compact parenchymal and vessel-associated amyloid deposits. The novel imaging platform offers new prospects for in vivo studies into Alzheimers disease mechanisms in animal models as well as longitudinal monitoring of therapeutic responses at a single plaque level.

neuroscience↗

bioRxiv2019

Tau deposition is associated with imaging patterns of tissue calcification in the P301L mouse model of human tauopathy

Brain calcification is associated with several neurodegenerative proteinopathies. Here, we report a new phenotype of intracranial calcification in transgenic P301L mice overexpressing 4 repeat tau. P301L mice (Thy1.2) of 3, 5, 9 and 18-25 months-of-age and age-matched non-transgenic littermates were assessed using in vivo/ex vivo magnetic resonance imaging (MRI) with a gradient recalled echo sequence and micro computed tomography (CT). Susceptibility weighted images computed from the gradient recalled echo data revealed regional hypointensities in the hippocampus, cortex, caudate nucleus and thalamus of P301L mice, which in corresponding phase images indicated diamagnetic lesions. Concomitantly, {micro}CT detected hyperdense lesions. Occurrence of diamagnetic susceptibility lesions in the hippocampus, increased with age. Immunochemical staining of brain sections revealed bone protein-positive deposits. Furthermore, intra-neuronal and vessel-associated protein-containing nodules co-localized with phosphorylated-tau (AT8 and AT100) in the hippocampus. Protein-containing nodules were detected also in the thalamus in the absence of phosphorylated-tau deposition. In contrast, osteocalcin-containing nodules were vessel-associated, indicating ossified vessels, in the thalamus in absence of phosphorylated-tau. In summary, MRI and {micro}CT demonstrated imaging pattern of intracranial calcification, concomitant with immunohistochemical evidence of formation of protein deposits containing bone proteins along with phosphorylated-tau in the P301L mouse model of human tauopathy. The P301L mouse model may thus serve as a future model to study the pathogenesis of brain calcifications in tauopathies.

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