Biology subjects

Tackenberg, C.

Publications and source records attributed to Tackenberg, C..

4 recordsLinked to original sources

bioRxiv2022

Molecular and anatomical roadmap of stroke pathology in immunodeficient mice

BackgroundStroke remains a leading cause of disability and death worldwide. It has become apparent that inflammation and immune mediators have a pre-dominant role in initial tissue damage and long-term recovery following the injury. Still, different immunosuppressed mouse models are necessary in stroke research e.g., to evaluate therapies using human cell grafts. Despite mounting evidence delineating the importance of inflammation in the stroke pathology, it is poorly described to what extent partial immune deficiency influences the overall stroke outcome. MethodsHere, we assessed the stroke pathology of popular genetic immunodeficient mouse models, i.e., NOD scid gamma (NSG) and recombination activating gene 2 (Rag2-/-) mice as well as pharmacologically immunosuppressed mice and compared them to immune competent, wildtype (WT) C57BL/6J mice up to three weeks after injury. We performed histology, gene expression profiling, serum analysis and functional behavioural tests to identify the impact of immunosuppression on the stroke progression. ResultsWe detected distinct changes in microglia infiltration, scar-forming and vascular repair in immune-suppressed mice three weeks after injury. Gene expression analysis of stroked tissue revealed the strongest deviation from immune competent mice was observed in NSG mice, for instance, affecting immunological and angiogenic pathways. Pharmacological immunosuppression resulted in the least variation in gene expression compared with the WT. Major differences have been further identified in the systemic inflammatory response following stroke acutely and three weeks following injury. These anatomical, genetic, and systemic changes did not affect functional deficits and recovery in a time course of three weeks. To determine whether the timing of immunosuppression after stroke is critical, we compared mice with acute and delayed pharmacological immunosuppression after stroke. Mice with a delayed immunosuppression (7d) after stroke showed increased inflammatory and scarring responses compared to animals acutely treated with tacrolimus, thus more closely resembling WT pathology. Transplantation of human cells in the brains of immunosuppressed mouse models led to prolonged cell survival in all immunosuppressed mouse models, which was most consistent in NSG and Rag2-/- mice. ConclusionsIn sum, we detected distinct anatomical and molecular changes in the stroke pathology between the individual immunosuppressed mouse models that should be carefully considered when selecting an appropriate mouse model for stroke research.

neuroscience↗

bioRxiv2022

Xeno-free induced pluripotent stem cell-derived neural progenitor cells for in vivo applications

Cell-based therapies are a promising treatment paradigm for neurodegenerative diseases and other brain injuries. Despite recent advances in stem cell technology, major concerns have been raised regarding the feasibility and safety of cell therapies for clinical applications. Here, we generate good manufacturing practice (GMP)-compatible neural progenitor cells (NPCs) from transgene- and xeno-free induced pluripotent stem cells (iPSCs) that can be smoothly adapted for clinical applications. The produced NPCs have a stable gene-expression over at least 15 passages and can be scaled for up to 1018 cells per initially seeded 106 cells. To ensure a pure NPC population for in vivo applications, we reduce risks of iPSC contamination using micro RNA-switch technology as a safety checkpoint. Using lentiviral transduction with a fluorescent and bioluminescent dual-reporter construct, combined with non-invasive in vivo bioluminescent imaging, we longitudinally tracked the grafted cells in healthy wild-type and genetically immunosuppressed mice as well as in a mouse model of ischemic stroke. Long term in-depth characterization revealed that transplanted cells have the capability to survive and spontaneously differentiate into functional and mature neurons throughout a time course of a month.

neuroscience↗

bioRxiv2021

Deep learning based behavioral profiling of rodent stroke recovery

Stroke research heavily relies on rodent behavior when assessing underlying disease mechanisms and treatment efficacy. Although functional motor recovery is considered the primary targeted outcome, tests in rodents are still poorly reproducible, and often unsuitable for unraveling the complex behavior after injury. Here, we provide a comprehensive 3D gait analysis of mice after focal cerebral ischemia based on the new deep learning-based software (DeepLabCut, DLC) that only requires basic behavioral equipment. We demonstrate a high precision 3D tracking of 10 body parts (including all relevant joints and reference landmarks) in several mouse strains with an accuracy of 99.4%. Building on this rigor motion tracking, a comprehensive post-analysis (with >100 parameters) unveils biologically relevant differences in locomotor profiles after a stroke over a time course of three weeks. We further refine the widely used ladder rung test using deep learning and compare its performance to human annotators. The generated DLC-assisted tests were then benchmarked to five widely used conventional behavioral set-ups (neurological scoring, rotarod, ladder rung walk, cylinder test, and single-pellet grasping) regarding sensitivity, accuracy, time use and costs. We conclude that deep learning-based motion tracking with comprehensive post-analysis provides accurate and sensitive data to describe the complex recovery of rodents following a stroke. The experimental set-up and analysis can also benefit a range of other neurological injuries that affect locomotion.

neuroscience↗

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↗