Biology subjects

Schwamborn, J. C.

Publications and source records attributed to Schwamborn, J. C..

32 records · Page 2Linked to original sources

bioRxiv2023

CLN3 deficiency leads to neurological and metabolic perturbations during early development

Juvenile Neuronal Ceroid Lipofuscinosis (or Batten disease) is an autosomal recessive, rare neurodegenerative disorder that affects mainly children above the age of 5 years and is most commonly caused by mutations in the highly conserved CLN3 gene. Here, we generated cln3 morphants and stable mutant lines in zebrafish. Although neither morphant nor mutant cln3 larvae showed any obvious developmental or morphological defects, behavioral phenotyping of the mutant larvae revealed higher basal activity, hyposensitivity to abrupt light changes and hypersensitivity to pro-convulsive drugs. Importantly, in-depth metabolomics and lipidomics analyses revealed significant accumulation of several glycerophosphodiesters (GPDs) and a global decrease of bis(monoacylglycero)phosphate (BMP) species, two classes of molecules previously proposed as potential biomarkers for CLN3 disease based on independent studies in other organisms. We could also demonstrate GPD accumulation in human-induced pluripotent stem cell-derived cerebral organoids carrying a pathogenic variant for CLN3. Our models revealed that GPDs accumulate at very early stages of life in the absence of functional CLN3 and highlight glycerophosphoinositol and BMP as promising biomarker candidates for pre-symptomatic CLN3 disease.

molecular biology↗

bioRxiv2022

Development of a multi-sensor integrated midbrain organoid-on-a-chip platform for studying Parkinson's disease

Due to its ability to recapitulate key pathological processes in vitro, midbrain organoid technology has significantly advanced the modeling of Parkinsons disease over the last few years. However, some limitations such as insufficient tissue differentiation and maturation, deficient nutrient supply, and low analytical accessibility persist, altogether restricting the technology from reaching its full potential. To overcome these drawbacks, we have developed a multi-sensor integrated organ-on-a-chip platform capable of monitoring the electrophysiological, respiratory, and dopaminergic activity of human midbrain organoids. Our study showed that microfluidic cultivation resulted in a marked reduction in necrotic core formation, improved tissue differentiation as well as the recapitulation of key pathological hallmarks. Non-invasive monitoring employing an orthogonal sensing strategy revealed a clear time dependency in the onset of Parkinsons disease-related phenotypes, reflecting the complex progression of the neurodegenerative disorder. Furthermore, drug-mediated rescue effects were observed after treatment with the repurposed compound 2-hydroxypropyl {beta}-cyclodextrin, highlighting the platforms potential in the context of drug screening applications as well as personalized medicine.

bioengineering↗

bioRxiv2022

Microglia integration into human midbrain organoids leads to increased neuronal maturation and functionality.

The human brain is a complex, three-dimensional structure. To better recapitulate brain complexity, recent efforts have focused on the development of human specific midbrain organoids. Human iPSC-derived midbrain organoids consist of differentiated and functional neurons, which contain active synapses, as well as astrocytes and oligodendrocytes. However, the absence of microglia, with their ability to remodel neuronal networks and phagocytose apoptotic cells and debris, represents a major disadvantage for the current midbrain organoid systems. Additionally, neuro-inflammation related disease modeling is not possible in the absence of microglia. So far, no studies about the effects of human iPSC-derived microglia on midbrain organoid neural cells have been published. Here we describe an approach to derive microglia from human iPSCs and integrate them into iPSC-derived midbrain organoids. Using single nuclear RNA Sequencing, we provide a detailed characterization of microglia in midbrain organoids as well as the influence of their presence on the other cells of the organoids. Furthermore, we describe the effects that microglia have on cell death and oxidative stress- related gene expression. Finally, we show that microglia in midbrain organoids affect synaptic remodeling and increase neuronal excitability. Altogether, we show a more suitable system to further investigate brain development, as well as neurodegenerative diseases and neuro- inflammation. Main Points- Macrophage precursors can be efficiently co-cultured with midbrain organoids, they integrate into the tissue and differentiate into microglia in 3D. - Organoids containing microglia have a smaller size and show a down-regulation of oxidative stress-related genes. - Organoids co-cultured with microglia show differences in genes related to synaptic remodeling and action potential, as well as a more spontaneous action potential firing.

neuroscience↗

bioRxiv2021

Synaptic decline precedes dopaminergic neuronal loss in human midbrain organoids harboring a triplication of the SNCA gene

1.Increased levels of the protein alpha-synuclein (-syn) are associated with the development of neurodegenerative diseases like Parkinsons disease (PD). In physiological conditions, -syn modulates synaptic plasticity, neurogenesis and neuronal survival. However, its pathogenic accumulation and aggregation results in toxicity and neurodegeneration. Here, we used a PD patient specific midbrain organoid model derived from induced pluripotent stem cells harboring a triplication in the SNCA gene to study PD-associated phenotypes. The model recapitulates the two main hallmarks of PD, which are -syn aggregation and loss of dopaminergic neurons. Additionally, impairments in astrocyte differentiation were detected. Transcriptomics data indicate that synaptic function is impaired in PD specific midbrain organoids. This is further confirmed by alterations in synapse number and electrophysiological activity. We found that synaptic decline precedes neurodegeneration. Finally, this study substantiates that patient specific midbrain organoids allow a personalized phenotyping, which make them an interesting tool for precision medicine and drug discovery.

neuroscience↗

bioRxiv2021

Mechanistic model-driven exometabolomic characterisation of human dopaminergic neuronal metabolism

Starting with a comprehensive generic reconstruction of human metabolism, we generated high-quality, constraint-based, genome-scale, cell-type and condition specific models of metabolism in human dopaminergic neurons, the cell type most vulnerable to degeneration in Parkinsons disease. They are a synthesis of extensive manual curation of the biochemical literature on neuronal metabolism, together with novel, quantitative, transcriptomic and targeted exometabolomic data from human stem cell-derived, midbrainspecific, dopaminergic neurons in vitro. Thermodynamic constraint-based modelling enabled qualitatively accurate and moderately quantitatively accurate prediction of dopaminergic neuronal metabolite exchange fluxes, including predicting the consequences of metabolic perturbations in a manner also consistent with literature on monogenic mitochondrial Parkinsons disease. These dopaminergic neurons models provide a foundation for a quantitative systems biochemistry approach to metabolic dysfunction in Parkinsons disease. Moreover, the plethora of novel mathematical and computational approaches required to develop them are generalisable to study any other disease associated with metabolic dysfunction.

systems biology↗

bioRxiv2021

Neuronal hyperactivity in a LRRK2-G2019S cellular model of Parkinson's Disease

Monogenic Parkinsons Disease can be caused by a mutation in the leucine-rich repeat kinase 2 (LRRK2) gene, causing a late-onset autosomal dominant inherited form of Parkinsons Disease. The function of the LRRK2 gene is incompletely understood, but several in vitro studies have reported that LRRK2-G2019S mutations affect neurite branching, calcium homeostasis and mitochondrial function, but thus far, there have been no reports of effects on electrophysiological activity. We assessed the neuronal activity of induced pluripotent stem cell derived neurons from Parkinsons Disease patients with LRRK2-G2019S mutations and isogenic controls. Neuronal activity of spontaneously firing neuronal populations was recorded with a fluorescent calcium-sensitive dye (Fluo-4) and analysed with a novel image analysis pipeline that combined semi-automated neuronal segmentation and quantification of calcium transient properties. Compared with controls, LRRK2-G2019S mutants have shortened inter-spike intervals and an increased rate of spontaneous calcium transient induction.

neuroscience↗

bioRxiv2020

Human skeletal muscle organoids model fetal myogenesis and sustain uncommitted PAX7 myogenic progenitors

In vitro culture systems that structurally model human myogenesis and promote PAX7+ myogenic progenitor maturation have not been established. Here we report that human skeletal muscle organoids can be differentiated from induced pluripotent stem cell lines to contain paraxial mesoderm and neuromesodermal progenitors and develop into organized structures reassembling neural plate border and dermomyotome. Culture conditions instigate neural lineage arrest and promote fetal hypaxial myogenesis towards limb axial anatomical identity, with generation of sustainable uncommitted PAX7 myogenic progenitors and fibroadipogenic (PDGFRa+) progenitor populations equivalent to those from the second trimester of human gestation. Single cell comparison to human fetal and adult myogenic progenitors reveals distinct molecular signatures for non-dividing myogenic progenitors in activated (CD44High/CD98+/MYOD1+) and dormant (PAX7High/FBN1High/SPRY1High) states. Our approach provides a robust 3D in vitro developmental system for investigating muscle tissue morphogenesis and homeostasis.

bioengineering↗

bioRxiv2020

Comparative analysis of human brain organoids of brainstem and midbrain at single-cell resolution

Human brain organoids provide us the means to investigate human brain development and neurological diseases, and single-cell RNA-sequencing (scRNA-seq) technologies allow us to identify homologous cell types and the molecular heterogeneity between individual cells. Previously established human brain organoids of brainstem (hBSOs) and midbrain (hMBOs) were analyzed by scRNA-seq, but the difference in cellular composition between these organoids remains unclear. Here, we integrated and compared the single-cell transcriptome of hBSOs and hMBOs. Our analysis demonstrated that the hBSOs and hMBOs contain some unique cell types, including inflammatory and mesenchymal cells. Further comparison of the hBSOs and hMBOs with publicly available scRNA-seq dataset of human fetal midbrain (hMB) showed high similarity in their neuronal components. These results provide new insights into human brain organoid technologies.

bioinformatics↗

bioRxiv2019

PARK7/DJ-1 promotes pyruvate dehydrogenase activity and maintains Treg homeostasis

Pyruvate dehydrogenase (PDH) is the gatekeeper enzyme into the tricarboxylic acid (TCA) cycle. Here we show that PARK7/DJ-1, a key familial Parkinsons disease (PD) gene, is a pacemaker controlling PDH activity in CD4 regulatory T cells (Tregs). DJ-1 bound to PDH-E1 beta (PDHB), inhibiting the phosphorylation of PDH-E1 alpha (PDHA), thus promoting PDH activity and oxidative phosphorylation (OXPHOS). Dj-1 depletion impaired Treg proliferation and cellularity maintenance in older mice, increasing the severity during the remission phase of experimental autoimmune encephalomyelitis (EAE). The compromised proliferation and differentiation of Tregs in Dj-1 knockout mice were caused via regulating PDH activity. These findings provide novel insight into the already complicated regulatory machinery of the PDH complex and demonstrate that the DJ-1-PDHB axis represents a potent target to maintain Treg homeostasis, which is dysregulated in many complex diseases.

biochemistry↗

bioRxiv2019

Cultivation and characterization of human midbrain organoids in sensor integrated microfluidic chips

1.With its ability to emulate microarchitectures and functional characteristics of native organs in vitro, induced pluripotent stem cell (iPSC) technology has enabled the generation of a plethora of organotypic constructs, including that of the human midbrain. However, reproducibly engineering and differentiating such human midbrain organoids (hMOs) under a biomimetic environment favorable for brain development still remains challenging. This study sets out to address this problem by combining the potential of iPSC technology with the advantages of microfluidics, namely its precise control over fluid flow combined with sensor integration. Here, we present a novel sensor-integrated platform for the long-term cultivation and non-invasive monitoring of hMOs under an interstitial flow regime. Our results show that dynamic cultivation of iPSC-derived hMOs maintains high cellular viabilities and dopaminergic neuron differentiation over prolonged cultivation periods of up to 50 days.

bioengineering↗

bioRxiv2019

Parkinson’s disease phenotypes in patient specific brain organoids are improved by HP-β-CD treatment

The etiology of Parkinsons disease (PD) is only partially understood despite the fact that environmental causes, risk factors, and specific gene mutations are contributors to the disease. Biallelic mutations in the PTEN-induced putative kinase 1 (PINK1) gene involved in mitochondrial homeostasis, vesicle trafficking, and autophagy, are sufficient to cause PD. By comparing PD patient-derived cells, we show differences in their energetic profile, imbalanced proliferation, apoptosis, mitophagy, and a reduced differentiation efficiency to dopaminergic neurons compared to control cells. Using CRISPR/Cas9 gene editing, correction of a patients point mutation ameliorated the metabolic properties and neuronal firing rates but without reversing the differentiation phenotype. However, treatment with 2-Hydroxypropyl-{beta}-Cyclodextrin (HP-{beta}-CD) increased the mitophagy capacity of neurons leading to an improved dopaminergic differentiation of patient specific neurons in midbrain organoids. In conclusion, we show that treatment with a repurposed compound is sufficient for restoring dopaminergic differentiation of PD patient-derived cells.

neuroscience↗

bioRxiv2019

Machine learning-assisted neurotoxicity prediction in human midbrain organoids

A major challenge in the field of neurodegenerative diseases is the poor translation of pre-clinical models to clinical applications. The human brain is an immensely complex structure, which makes it difficult to recapitulate its development, function and disorders. In the recent years, brain organoids derived from human induced pluripotent stem cells have risen as novel tools to study neurodegenerative diseases such as Parkinsons disease (PD). PD is a multifactorial disorder, with aging, genetics and environmental factors as key etiological elements. The majority of the PD cases are idiopathic and proposed to result from a complex interaction between genetic predisposition and environmental exposure. Consequently, the identification of potentially disease causing environmental factors is of critical importance. Organoids, as complex multi-cellular tissue proxies, are an ideal tool to study cellular response to environmental changes. However, with increasing complexity of the system, usage of quantitative tools becomes challenging. This led us to develop an automated high-content image analysis pipeline for image-based cell profiling in the organoid system. Here, we introduce a midbrain organoid system that recapitulates features of neurotoxin-induced PD, representing a platform for machine-learning-assisted prediction of neurotoxicity in high-content imaging data. This model is a valuable tool for advanced in vitro PD modeling and for the screening of putative neurotoxic compounds.

pharmacology and toxicology↗

bioRxiv2018

Automated high-throughput high-content autophagy and mitophagy analysis platform

Autophagy and mitophagy play a central role in cellular homeostasis. In pathological conditions, the flow of autophagy and mitophagy can be affected at multiple and distinct steps of the pathways. Unfortunately, the level of detail of current state of the art analyses does not allow detection or dissection of pathway intermediates. Moreover, is conducted in low-throughput manner on bulk cell populations. Defining autophagy and mitophagy pathway intermediates in a high-throughput manner is technologically challenging, and has not been addressed so far. Here, we overcome those limitations and developed a novel high-throughput phenotyping platform with automated high-content image analysis to assess autophagy and mitophagy pathway intermediates.

cell biology↗

bioRxiv2017

In Vivo Phenotyping Of Parkinson-Specific Stem Cells Reveals Increased a-Synuclein Levels But No Spreading

Parkinson's disease is a progressive age-associated neurological disorder. One of the major neuropathological hallmarks of Parkinsons disease is the appearance of protein aggregates, mainly consisting of the protein alpha-Synuclein. These aggregates have been described both in genetic as well as idiopathic forms of the disease. Currently, Parkinsons disease patient-specific induced pluripotent stem cells (iPSCs) are mainly used for in vitro disease modeling or for experimental cell replacement approaches. Here, we demonstrate that these cells can be used for in vivo disease modeling. We show that Parkinsons disease patient-specific, iPSC-derived neurons carrying the LRRK2-G2019S mutation show an upregulation of alpha-Synuclein after transplantation in the mouse brain. However, further investigations indicate that the increased human alpha-Synuclein levels fail to induce spreading or aggregation in the mouse brain. We therefore conclude that grafting of these cells into the mouse brain is suitable for cell autonomous in vivo disease modeling but has strong limitations beyond that. Furthermore, our results support the hypothesis that there might be a species barrier between human to mouse concerning alpha-Synuclein spreading.

neuroscience↗