Identification and development of TRPM4 antagonists to counteract neuronal excitotoxicity.

Neurodegeneration in central nervous system disorders is linked to dysregulated neuronal calcium. Direct inhibition of glutamate-induced neuronal calcium influx, particularly via N-methyl-D-aspartate receptors (NMDAR), has led to adverse effects and clinical trial failures. A more feasible approach is to modulate NMDAR activity or calcium signaling indirectly.

Generation of two isogenic human iPSC lines (ZIPi013-B-1, ZIPi013-B-2) carrying a CRISPR/Cas9-mediated deletion of TRPM4.

Two isogenic hiPSC lines, ZIPi013-B-1 and ZIPi013-B-2, were generated by CRISPR/Cas9-mediated indels in the TRPM4 gene of the previously published ZIPi013-B. TRPM4 belongs to the evolutionarily conserved family of transient receptor potential (TRP) channels. It is expressed ubiquitously and its activity is regulated by intracellular calcium binding, changes in membrane potential, phosphoinositide lipids in the plasma membrane and the local concentration of cytoplasmic ATP and ADP.

The NR4A2/VGF pathway fuels inflammation-induced neurodegeneration via promoting neuronal glycolysis.

A disturbed balance between excitation and inhibition (E/I balance) is increasingly recognized as a key driver of neurodegeneration in multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system. To understand how chronic hyperexcitability contributes to neuronal loss in MS, we transcriptionally profiled neurons from mice lacking inhibitory metabotropic glutamate signaling with shifted E/I balance and increased vulnerability to inflammation-induced neurodegeneration. This revealed a prominent induction of the nuclear receptor NR4A2 in neurons.

MicroRNA-92a-CPEB3 axis protects neurons against inflammatory neurodegeneration.

Neuroinflammation causes neuronal injury in multiple sclerosis (MS) and other neurological diseases. MicroRNAs (miRNAs) are important modulators of neuronal stress responses, but knowledge about their contribution to neuronal protection or damage during inflammation is limited. Here, we constructed a regulatory miRNA-mRNA network of inflamed motor neurons by leveraging cell type-specific miRNA and mRNA sequencing of mice undergoing experimental autoimmune encephalomyelitis (EAE).

Human isogenic cells of the neurovascular unit exert transcriptomic cell type-specific effects on a blood-brain barrier in vitro model of late-onset Alzheimer disease.

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.

Generation of a set of induced pluripotent stem cell lines from two Alzheimer disease patients carrying APOE4 (MLUi007-J; MLUi008-A) and healthy old donors carrying APOE3 (MLUi009-A; MLUi010-B) to study APOE in aging and disease.

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).

The mechanism of macroautophagy: The movie.

This animated movie presents the mechanism of macroautophagy, hereafter autophagy, by showing the molecular features of the formation of autophagosomes, the hallmark organelle of this intracellular catabolic pathway. It is based on our current knowledge and it also illustrates how autophagosomes can recognize and eliminate selected cargoes.

High-content screening of mitochondrial polarization in neural cells derived from human pluripotent stem cells.

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.

G9a dictates neuronal vulnerability to inflammatory stress via transcriptional control of ferroptosis.

Neuroinflammation leads to neuronal stress responses that contribute to neuronal dysfunction and loss. However, treatments that stabilize neurons and prevent their destruction are still lacking. Here, we identify the histone methyltransferase G9a as a druggable epigenetic regulator of neuronal vulnerability to inflammation.

A hybrid approach unveils drug repurposing candidates targeting an Alzheimer pathophysiology mechanism.

The high number of failed pre-clinical and clinical studies for compounds targeting Alzheimer disease (AD) has demonstrated that there is a need to reassess existing strategies. Here, we pursue a holistic, mechanism-centric drug repurposing approach combining computational analytics and experimental screening data. Based on this integrative workflow, we identified 77 druggable modifiers of tau phosphorylation (pTau).

The blood-brain barrier is dysregulated in COVID-19 and serves as a CNS entry route for SARS-CoV-2.

Neurological complications are common in COVID-19. Although SARS-CoV-2 has been detected in patients' brain tissues, its entry routes and resulting consequences are not well understood. Here, we show a pronounced upregulation of interferon signaling pathways of the neurovascular unit in fatal COVID-19.

Culturing human iPSC-derived neural progenitor cells on nanowire arrays: mapping the impact of nanowire length and array pitch on proliferation, viability, and membrane deformation.

Nanowire arrays used as cell culture substrates build a potent tool for advanced biological applications such as cargo delivery and biosensing. The unique topography of nanowire arrays, however, renders them a challenging growth environment for cells and explains why only basic cell lines have been employed in existing studies. Here, we present the culturing of human induced pluripotent stem cell-derived neural progenitor cells on rectangularly arranged nanowire arrays: In detail, we mapped the impact on proliferation, viability, and topography-induced membrane deformation across a multitude of array pitches (1, 3, 5, 10 μm) and nanowire lengths (1.

Droplet-based vitrification of adherent human induced pluripotent stem cells on alginate microcarrier influenced by adhesion time and matrix elasticity.

The gold standard in cryopreservation is still conventional slow freezing of single cells or small aggregates in suspension, although major cell loss and limitation to non-specialised cell types in stem cell technology are known drawbacks. The requirement for rapidly available therapeutic and diagnostic cell types is increasing constantly. In the case of human induced pluripotent stem cells (hiPSCs) or their derivates, more sophisticated cryopreservation protocols are needed to address this demand.

Targeting autophagy in disease: established and new strategies.

Macroautophagy/autophagy is an evolutionarily conserved pathway responsible for clearing cytosolic aggregated proteins, damaged organelles or invading microorganisms. Dysfunctional autophagy leads to pathological accumulation of the cargo, which has been linked to a range of human diseases, including neurodegenerative diseases, infectious and autoimmune diseases and various forms of cancer. Cumulative work in animal models, application of genetic tools and pharmacologically active compounds, has suggested the potential therapeutic value of autophagy modulation in disease, as diverse as Huntington, infection, or pancreatic cancer.

Enhancing mitochondrial activity in neurons protects against neurodegeneration in a mouse model of multiple sclerosis.

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).

Human iPSC-Derived Blood-Brain Barrier Models: Valuable Tools for Preclinical Drug Discovery and Development?

Translating basic biological knowledge into applications remains a key issue for effectively tackling neurodegenerative, neuroinflammatory, or neuroendocrine disorders. Efficient delivery of therapeutics across the neuroprotective blood-brain barrier (BBB) still poses a demanding challenge for drug development targeting central nervous system diseases. Validated in vitro models of the BBB could facilitate effective testing of drug candidates targeting the brain early in the drug discovery process during lead generation.

An automated and high-throughput-screening compatible pluripotent stem cell-based test platform for developmental and reproductive toxicity assessment of small molecule compounds.

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.

Lipid Mediator Profiles Predict Response to Therapy with an Oral Frankincense Extract in Relapsing-Remitting Multiple Sclerosis.

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.

Interfacing human induced pluripotent stem cell-derived neurons with designed nanowire arrays as a future platform for medical applications.

Nanostructured substrates such as nanowire arrays form a powerful tool for building next-generation medical devices. So far, human pluripotent stem cell-derived neurons-a revolutionary tool for studying physiological function and modeling neurodegenerative diseases-have not been applied to such innovative substrates, due to the highly demanding nature of stem cell quality control and directed differentiation procedures to generate specialized cell types. Our study closes this gap, by presenting electrophysiologically mature human pluripotent stem cell-derived neurons on a set of nanowires in different patterns and growth densities after only four weeks of maturation-thereof 14 to 16 days on the nanowire arrays.

Automated real-time monitoring of human pluripotent stem cell aggregation in stirred tank reactors.

The culture of human induced pluripotent stem cells (hiPSCs) at large scale becomes feasible with the aid of scalable suspension setups in continuously stirred tank reactors (CSTRs). Innovative monitoring options and emerging automated process control strategies allow for the necessary highly defined culture conditions. Next to standard process characteristics such as oxygen consumption, pH, and metabolite turnover, a reproducible and steady formation of hiPSC aggregates is vital for process scalability.

Generation of two human isogenic iPSC lines from fetal dermal fibroblasts.

Two isogenic hiPSC lines, ZIPi013-B and ZIPi013-E, were generated by reprogramming fetal dermal fibroblasts with episomal vectors. Previously, the same fetal fibroblasts were reprogrammed multiple times in a study comparing other reprogramming methods. As a consequence, the genomes have been sequenced multiple times.

Driving next-generation autophagy researchers towards translation (DRIVE), an international PhD training program on autophagy.

The European autophagy consortium (DRIVE) held its kick-off meeting in Groningen on the 14 and 15 of June 2018. This Marie Skłodowska-Curie Early Training Network was approved under the European Union's Horizon 2020 Research and Innovation Program and is funded for 4 years. Within DRIVE, 14 European research teams from academia and industry will train 15 PhD students through applied, cross-disciplinary and collaborative macroautophagy/autophagy research.