While transcripts of neuronal mitochondrial genes are strongly suppressed in central nervous system inflammation, it is unknown whether this results in mitochondrial dysfunction and whether an ...increase of mitochondrial function can rescue neurodegeneration. Here, we show that predominantly genes of the electron transport chain are suppressed in inflamed mouse neurons, resulting in impaired mitochondrial complex IV activity. This was associated with post-translational inactivation of the transcriptional co-regulator proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). In mice, neuronal overexpression of
, which encodes for PGC-1α, led to increased numbers of mitochondria, complex IV activity, and maximum respiratory capacity. Moreover,
-overexpressing neurons showed a higher mitochondrial membrane potential that related to an improved calcium buffering capacity. Accordingly, neuronal deletion of
aggravated neurodegeneration during experimental autoimmune encephalomyelitis, while neuronal overexpression of
ameliorated it. Our study provides systemic insights into mitochondrial dysfunction in neurons during inflammation and commends elevation of mitochondrial activity as a promising neuroprotective strategy.
Lipid mediators (LMs) are a unique class of immunoregulatory signalling molecules and known to be affected by frankincense extracts. We performed LM profiling by metabololipidomics in plasma samples ...from 28 relapsing-remitting multiple sclerosis (RR-MS) patients who took a standardised frankincense extract (SFE) daily for eight months in a clinical phase IIa trial (NCT01450124) and in 28 age- and gender-matched healthy controls. Magnetic resonance imaging, immunological outcomes and serum neurofilament light chain levels were correlated to changes in the LM profiles of the RR-MS cohort. Eight out of 44 analysed LMs were significantly reduced during an eight-month treatment period by the SFE and seven of these eight significant LM derive from the 5-lipoxygenase (5-LO) pathway. Baseline levels of 12- and 15-LO products were elevated in patients who exhibited disease activity (EDA) during SFE treatment compared to no-evidence-of-disease-activity (NEDA) patients and could predict treatment response to the SFE in a prediction model at baseline. Oral treatment with an SFE significantly reduces 5-LO-derived LMs in RR-MS patients during an eight-month treatment period. Treatment response to an SFE, however, seems to be related to 12-,15-LO and cyclooxygenase product levels before SFE exposure. Further studies should confirm their biomarker potential in RR-MS and SFE treatment.
Primary progressive multiple sclerosis (PPMS) shows a highly variable disease progression with poor prognosis and a characteristic accumulation of disabilities in patients. These hallmarks of PPMS ...make it difficult to diagnose and currently impossible to efficiently treat. This study aimed to identify plasma metabolite profiles that allow diagnosis of PPMS and its differentiation from the relapsing-remitting subtype (RRMS), primary neurodegenerative disease (Parkinson's disease, PD), and healthy controls (HCs) and that significantly change during the disease course and could serve as surrogate markers of multiple sclerosis (MS)-associated neurodegeneration over time. We applied untargeted high-resolution metabolomics to plasma samples to identify PPMS-specific signatures, validated our findings in independent sex- and age-matched PPMS and HC cohorts and built discriminatory models by partial least square discriminant analysis (PLS-DA). This signature was compared to sex- and age-matched RRMS patients, to patients with PD and HC. Finally, we investigated these metabolites in a longitudinal cohort of PPMS patients over a 24-month period. PLS-DA yielded predictive models for classification along with a set of 20 PPMS-specific informative metabolite markers. These metabolites suggest disease-specific alterations in glycerophospholipid and linoleic acid pathways. Notably, the glycerophospholipid LysoPC(20:0) significantly decreased during the observation period. These findings show potential for diagnosis and disease course monitoring, and might serve as biomarkers to assess treatment efficacy in future clinical trials for neuroprotective MS therapies.
While a link between inflammation and the development of neuropsychiatric disorders, including major depressive disorder (MDD) is supported by a growing body of evidence, little is known about the ...contribution of aberrant adaptive immunity in this context. Here, we conducted in-depth characterization of T cell phenotype and T cell receptor (TCR) repertoire in MDD. For this cross-sectional case-control study, we recruited antidepressant-free patients with MDD without any somatic or psychiatric comorbidities (
= 20), who were individually matched for sex, age, body mass index, and smoking status to a non-depressed control subject (
= 20). T cell phenotype and repertoire were interrogated using a combination of flow cytometry, gene expression analysis, and next generation sequencing. T cells from MDD patients showed significantly lower surface expression of the chemokine receptors CXCR3 and CCR6, which are known to be central to T cell differentiation and trafficking. In addition, we observed a shift within the CD4
T cell compartment characterized by a higher frequency of CD4
CD25
CD127
cells and higher
mRNA expression in purified CD4
T cells obtained from patients with MDD. Finally, flow cytometry-based TCR Vβ repertoire analysis indicated a less diverse CD4
T cell repertoire in MDD, which was corroborated by next generation sequencing of the TCR β chain CDR3 region. Overall, these results suggest that T cell phenotype and TCR utilization are skewed on several levels in patients with MDD. Our study identifies putative cellular and molecular signatures of dysregulated adaptive immunity and reinforces the notion that T cells are a pathophysiologically relevant cell population in this disorder.
Gene therapy is a promising option for severe forms of genetic diseases. We previously provided evidence for the feasibility of trans-splicing, exon skipping, and gene replacement in a mouse model of ...hypertrophic cardiomyopathy (HCM) carrying a mutation in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C). Here we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from an HCM patient carrying a heterozygous c.1358-1359insC MYBPC3 mutation and from a healthy donor. HCM hiPSC-CMs exhibited ∼50% lower MYBPC3 mRNA and cMyBP-C protein levels than control, no truncated cMyBP-C, larger cell size, and altered gene expression, thus reproducing human HCM features. We evaluated RNA trans-splicing and gene replacement after transducing hiPSC-CMs with adeno-associated virus. trans-splicing with 5′ or 3′ pre-trans-splicing molecules represented ∼1% of total MYBPC3 transcripts in healthy hiPSC-CMs. In contrast, gene replacement with the full-length MYBPC3 cDNA resulted in ∼2.5-fold higher MYBPC3 mRNA levels in HCM and control hiPSC-CMs. This restored the cMyBP-C level to 81% of the control level, suppressed hypertrophy, and partially restored gene expression to control level in HCM cells. This study provides evidence for (1) the feasibility of trans-splicing, although with low efficiency, and (2) efficient gene replacement in hiPSC-CMs with a MYBPC3 mutation.
Late-onset Alzheimer disease (LOAD) is the most frequent neurodegenerative disease, and the APOE ε4 allele is the most prominent risk factor for LOAD. Four human induced pluripotent stem cell (iPSC) ...lines MLUi007-J, MLUi008-B, MLUi009-A, and MLUi010-B were generated from LOAD patients and healthy matched donors by reprogramming of B-lymphoblastoid cells (B-LCLs) with episomal plasmids. The application of B-LCLs holds a great promise to model LOAD and other diseases because they can easily be generated from primary peripheral blood mononuclear cells (PBMCs) by infection with the Epstein-Barr virus (EBV).
We present a high-content screening (HCS) protocol for quantifying mitochondrial activity in live neural cells from human induced pluripotent stem cells (iPSCs). The assessment is based on ...mitochondrial membrane potential, which is influenced by the efficiency of mitochondrial bioenergetics. We describe how to perform the analysis using both an HCS platform and the open-source software CellProfiler. The protocol can identify the mitochondrial fitness of human neurons and may be used to carry out high-throughput compound screenings in patient-derived neural cells.
For complete details on the use and execution of this protocol, please refer to Lorenz et al. (2017) and Zink et al. (2020).
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•High-content screening (HCS) of live neural cells derived from human iPSCs•Assessment of mitochondrial activity based on mitochondrial membrane potential (MMP)•Quantification using automated microscopic image analysis•Data analysis using both an HCS system and the open-source software CellProfiler
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
We present a high-content screening (HCS) protocol for quantifying mitochondrial activity in live neural cells from human induced pluripotent stem cells (iPSCs). The assessment is based on mitochondrial membrane potential, which is influenced by the efficiency of mitochondrial bioenergetics. We describe how to perform the analysis using both an HCS platform and the open-source software CellProfiler. The protocol can identify the mitochondrial fitness of human neurons and may be used to carry out high-throughput compound screenings in patient-derived neural cells.
Background The function of the blood-brain barrier (BBB) is impaired in late-onset Alzheimer disease (LOAD), but the associated molecular mechanisms, particularly with respect to the high-risk ...APOE4/4 genotype, are not well understood. For this purpose, we developed a multicellular isogenic model of the neurovascular unit (NVU) based on human induced pluripotent stem cells. Methods The human NVU was modeled in vitro using isogenic co-cultures of astrocytes, brain capillary endothelial-like cells (BCECs), microglia-like cells, neural stem cells (NSCs), and pericytes. Physiological and pathophysiological properties were investigated as well as the influence of each single cell type on the characteristics and function of BCECs. The barriers established by BCECs were analyzed for specific gene transcription using high-throughput quantitative PCR. Results Co-cultures were found to tighten the barrier of BCECs and alter its transcriptomic profile under both healthy and disease conditions. In vitro differentiation of brain cell types that constitute the NVU was not affected by the LOAD background. The supportive effect of NSCs on the barrier established by BCECs was diminished under LOAD conditions. Transcriptomes of LOAD BCECs were modulated by different brain cell types. NSCs were found to have the strongest effect on BCEC gene regulation and maintenance of the BBB. Co-cultures showed cell type-specific functional contributions to BBB integrity under healthy and LOAD conditions. Conclusions Cell type-dependent transcriptional effects on LOAD BCECs were identified. Our study suggests that different brain cell types of the NVU have unique roles in maintaining barrier integrity that vary under healthy and LOAD conditions. Graphical abstract . Keywords: Apolipoprotein E, Human induced pluripotent cells, Blood-brain barrier, Neurovascular unit, Alzheimer disease, Late-onset Alzheimer disease
The embryonic stem cell test (EST) represents the only validated and accepted in vitro system for the detection and classification of compounds according to their developmental and reproductive ...teratogenic potency. The widespread implementation of the EST, however, in particular for routine application in pharmaceutical development, has not been achieved so far. Several drawbacks still limit the high-throughput screening of potential drug candidates in this format: The long assay period, the use of non-homogeneous viability assays, the low throughput analysis of marker protein expression and the compatibility of the assay procedures to automation. We have therefore introduced several advancements into the EST workflow: A reduction of the assay period, an introduction of homogeneous viability assays, and a straightforward analysis of marker proteins by flow cytometry and high content imaging to assess the impact of small molecules on differentiation capacity. Most importantly, essential parts of the assay procedure have been adapted to lab automation in 96-well format, thus enabling the interrogation of several compounds in parallel. In addition, extensive investigations were performed to explore the predictive capacity of this next-generation EST, by testing a set of well-known embryotoxicants that encompasses the full range of chemical-inherent embryotoxic potencies possible. Due to these significant improvements, the augmented workflow provides a basis for a sensitive, more rapid, and reproducible high throughput screening compatible platform to predict in vivo developmental toxicity from in vitro data which paves the road towards application in an industrial setting.
Graphical abstract
•The embryonic stem cell test to predict teratogenicity was made automation-compatible.
•Several key improvements to the assay procedure have been introduced to increase performance.
•The workflow was adapted to human iPS cells and isogenic fibroblast donor cells.
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