intracerebral-transplantation-vivo-bioluminescence-tracking-human

Intracerebral Transplantation and <em>In Vivo</em> Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point ...

University of Zurich

University of Zurich and ETH Zurich

Mitochondrial dysfunction is a prominent feature of Alzheimer's disease (AD) and increased production of reactive oxygen species (ROS) has been described in postmortem brain samples and animal models. However, these observations were made at a late stage of disease and the inability to examine an early, presymptomatic phase in human neurons impeded our understanding of cause or consequence of mitochondrial dysfunction in AD. We used human induced pluripotent stem cell-derived neuronal cells (iN cells) from sporadic AD (SAD) patients and healthy control subjects (HCS) to show aberrant mitochondrial function in patient-derived cells. We observed that neuronal cultures from some patients produced more ROS and displayed higher levels of DNA damage. Furthermore, patient-derived cells showed increased levels of oxidative phosphorylation chain complexes, whereas mitochondrial fission and fusion proteins were not affected. Surprisingly, these effects neither correlated with Aβ nor phosphorylated and total tau levels. Synaptic protein levels were also unaffected in SAD iN cells. The results of this study give new insights into constitutional metabolic changes in neurons from subjects prone to develop Alzheimer's pathology. They suggest that increased ROS production may have an integral role in the development of sporadic AD prior to the appearance of amyloid and tau pathology.

Oxidative stress and altered mitochondrial protein expression in the absence of amyloid-β and tau pathology in iPSC-derived neurons from sporadic Alzheimer's disease patients.

The amyloid precursor protein (APP) is endoproteolytically processed to generate either the neurotoxic beta-amyloid peptide (Aβ) or the secreted ectodomain APP alpha (sAPPα). While neurotrophic properties of sAPPα were suggested in several studies, it is still unclear if and how sAPPα counteracts pathogenic effects of Aβ. Direct comparisons with sAPPβ, produced in the Aβ-generating pathway, are missing in order to determine the role of sAPPα's carbonyl-terminal end in its possible neuroprotective activity.

The secreted APP ectodomain sAPPα, but not sAPPβ, protects neurons against Aβ oligomer-induced dendritic spine loss and increased tau phosphorylation.

Since the discovery of the induced pluripotent stem cell (iPSC) technique more than a decade ago, extensive progress has been made to develop clinically relevant cell culture systems. Alzheimer's disease (AD) is the most common neurodegenerative disease, accounting for approximately two thirds of all cases of dementia. The massively increasing number of affected individuals explains the major interest of research in this disease as well as the strong need for better understanding of disease mechanisms.

Alzheimer's in a dish - induced pluripotent stem cell-based disease modeling.

The distinct organization of the brain's vasculature ensures the adequate delivery of oxygen and nutrients during development and adulthood. Acute and chronic pathological changes of the vascular system have been implicated in many neurological disorders including stroke and dementia. Here, we describe a fast, automated method that allows the highly reproducible, quantitative assessment of distinct vascular parameters and their changes based on the open source software Fiji (ImageJ). In particular, we developed a practical guide to reliably measure aspects of growth, repair and maturation of the brain's vasculature during development and neurovascular disease in mice and humans. The script can be used to assess the effects of different external factors including pharmacological treatments or disease states. Moreover, the procedure is expandable to blood vessels of other organs and vascular  models.

A Practical Guide to the Automated Analysis of Vascular Growth, Maturation and Injury in the Brain.

Familial forms of Alzheimer's disease (AD) are caused by mutations in the presenilin genes or in the gene encoding for the amyloid precursor protein (APP). Proteolytic cleavage of APP generates the β-amyloid peptide (Aβ), which aggregates into amyloid plaques, one of the major hallmarks of AD. APP mutations within the Aβ sequence, so-called intra-Aβ mutations, cluster around position E693 of APP, which corresponds to position E22 in the Aβ sequence. One of these mutations is the Osaka mutation, E693Δ, which has unique aggregation properties with patients showing unusually low brain amyloid levels on amyloid PET scans. Despite intense research on the pathomechanisms of different intra-Aβ mutants, our knowledge is limited due to controversial findings in various studies. Here, we investigated in an ex vivo experimental system the neuro- and synaptotoxic properties of two intra-Aβ mutants with different intrinsic aggregation propensities, the Osaka mutation E22Δ and the Arctic mutation E22G, and compared them to wild-type (wt) Aβ. Experiments in hippocampal slice cultures from transgenic mice were complemented by treating wild-type slices with recombinantly produced Aβ40 or Aβ42 containing the respective intra-Aβ mutations. Our analyses revealed that wt Aβ and E22G Aβ, both recombinant and transgenic, caused a loss of dendritic spines along with an increase in tau phosphorylation and tau-dependent neurodegeneration. In all experiments, the 42-residue variants of wt and E22G Aβ showed stronger effects than the respective Aβ40 isoforms. In contrast, E22Δ Aβ neither reduced dendritic spine density nor resulted in increased tau phosphorylation or neuronal cell death in our ex vivo system. Our findings suggest that the previously reported major differences in the aggregation kinetics between E22G and E22Δ Aβ are likely reflected in different disease pathomechanisms.

Familial Alzheimer's disease mutations at position 22 of the amyloid β-peptide sequence differentially affect synaptic loss, tau phosphorylation and neuronal cell death in an ex vivo system.

Blood brain barrier (BBB) damage is an important pathophysiological feature of ischemic stroke which significantly contributes to development of severe brain injury and therefore is an interesting target for therapeutic intervention. A popular permanent occlusion model to study long term recovery following stroke is the photothrombotic model, which so far has not been anatomically characterized for BBB leakage beyond the acute phase. Here, we observed enhanced BBB permeability over a time course of 3 weeks in peri-infarct and core regions of the ischemic cortex. Slight increases in BBB permeability could also be seen in the contralesional cortex, hippocampus and the cerebellum at different time points, regions where lesion-induced degeneration of pathways is prominent. Severe damage of tight and adherens junctions and loss of pericytes was observed within the peri-infarct region. Overall, the photothrombotic stroke model reproduces a variety of features observed in human stroke and thus, represents a suitable model to study BBB damage and therapeutic approaches interfering with this process.

Characterization of the Blood Brain Barrier Disruption in the Photothrombotic Stroke Model.

Induced pluripotent stem cells (iPSCs) can be differentiated into virtually every desired cell type, offering significant potential for modeling human diseases in vitro. A disadvantage is that iPSC-derived cells represent an immature, which presents a major limitation for modeling age-related diseases such as Alzheimer's disease. Evidence suggests that culturing iPSC neurons in a 3D environment may increase neuronal maturity. However, current 3D cell culture systems are cumbersome and time-consuming.

Increased maturation of iPSC-derived neurons in a hydrogel-based 3D culture.

The apolipoprotein E4 (APOE4) variant is the strongest genetic risk factor for Alzheimer disease (AD), while the APOE2 allele is protective. A major question is how different APOE genotypes affect the physiology of astrocytes, the main APOE-producing brain cells. Here, we differentiated human APOE-isogenic induced pluripotent stem cells (iPSCs) (APOE4, E3, E2, and APOE knockout [APOE-KO]) to functional "iAstrocytes". Mass-spectrometry-based proteomic analysis showed genotype-dependent reductions of cholesterol and lipid metabolic and biosynthetic pathways (reduction: APOE4 >E3 >E2). Cholesterol efflux and biosynthesis were reduced in APOE4 iAstrocytes, while subcellular localization of cholesterol in lysosomes was elevated. An increase in immunoregulatory proteomic pathways (APOE4 >E3 >E2) was accompanied by elevated cytokine release in APOE4 cells (APOE4 >E3 >E2 >KO). Activation of iAstrocytes exacerbated proteomic changes and cytokine secretion mostly in APOE4 iAstrocytes, while APOE2 and APOE-KO iAstrocytes were least affected. Taken together, APOE4 iAstrocytes reveal a disease-relevant phenotype, causing dysregulated cholesterol/lipid homeostasis, increased inflammatory signaling, and reduced β-amyloid uptake, while APOE2 iAstrocytes show opposing effects.

Article	Year
Oxidative stress and altered mitochondrial protein expression in the absence of amyloid-β and tau pathology in iPSC-derived neurons from sporadic Alzheimer's disease patients. Stem cell research\| PubMed ID: 29414602	2018
The secreted APP ectodomain sAPPα, but not sAPPβ, protects neurons against Aβ oligomer-induced dendritic spine loss and increased tau phosphorylation. Molecular brain\| PubMed ID: 30922360	2019
Alzheimer's in a dish - induced pluripotent stem cell-based disease modeling. Translational neurodegeneration\| PubMed ID: 31338163	2019
A Practical Guide to the Automated Analysis of Vascular Growth, Maturation and Injury in the Brain. Frontiers in neuroscience\| PubMed ID: 32265643	2020
Familial Alzheimer's disease mutations at position 22 of the amyloid β-peptide sequence differentially affect synaptic loss, tau phosphorylation and neuronal cell death in an ex vivo system. PloS one\| PubMed ID: 32966331	2020
Characterization of the Blood Brain Barrier Disruption in the Photothrombotic Stroke Model. Frontiers in physiology\| PubMed ID: 33262704	2020
Increased maturation of iPSC-derived neurons in a hydrogel-based 3D culture. Journal of neuroscience methods\| PubMed ID: 34126141	2021
APOE2, E3, and E4 differentially modulate cellular homeostasis, cholesterol metabolism, and inflammatory response in isogenic iPSC-derived astrocytes. Stem cell reports\| PubMed ID: 34919811	2022

Christian Tackenberg

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