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. Mechanistically, NR4A2 increased susceptibility to excitotoxicity by stimulating continuous VGF secretion leading to glycolysis-dependent neuronal cell death. Extending these findings to people with MS (pwMS), we observed increased VGF levels in serum and brain biopsies. Notably, neuron-specific deletion of Vgf in a mouse model of MS ameliorated neurodegeneration. These findings underscore the detrimental effect of a persistent metabolic shift driven by excitatory activity as a fundamental mechanism in inflammation-induced neurodegeneration.

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. 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. .

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). We found robust induction of miR-92a in inflamed spinal cord neurons and identified cytoplasmic polyadenylation element-binding protein 3 (Cpeb3) as a key target of miR-92a-mediated posttranscriptional silencing. We detected CPEB3 repression in inflamed neurons in murine EAE and human MS. Moreover, both miR-92a delivery and Cpeb3 deletion protected neuronal cultures against excitotoxicity. Supporting a detrimental effect of Cpeb3 in vivo, neuron-specific deletion in conditional Cpeb3 knockout animals led to reduced inflammation-induced clinical disability in EAE. Together, we identified a neuroprotective miR-92a-Cpeb3 axis in neuroinflammation that might serve as potential treatment target to limit inflammation-induced neuronal damage.

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).

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. 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).

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. In murine experimental autoimmune encephalomyelitis (EAE) and human multiple sclerosis (MS), we found that the G9a-catalyzed repressive epigenetic mark H3K9me2 was robustly induced by neuroinflammation. G9a activity repressed anti-ferroptotic genes, diminished intracellular glutathione levels, and triggered the iron-dependent programmed cell death pathway ferroptosis. Conversely, pharmacological treatment of EAE mice with a G9a inhibitor restored anti-ferroptotic gene expression, reduced inflammation-induced neuronal loss, and improved clinical outcome. Similarly, neuronal anti-ferroptotic gene expression was reduced in MS brain tissue and was boosted by G9a inhibition in human neuronal cultures. This study identifies G9a as a critical transcriptional enhancer of neuronal ferroptosis and potential therapeutic target to counteract inflammation-induced neurodegeneration.

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). One of the upstream modulators of pTau, HDAC6, was screened with 5,632 drugs in a tau-specific assay, resulting in the identification of 20 repurposing candidates. Four compounds and their known targets were found to have a link to AD-specific genes. Our approach can be applied to a variety of AD-associated pathophysiological mechanisms to identify more repurposing candidates.

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. By investigating the susceptibility of human induced pluripotent stem cell (hiPSC)-derived brain capillary endothelial-like cells (BCECs) to SARS-CoV-2 infection, we found that BCECs were infected and recapitulated transcriptional changes detected in vivo. While BCECs were not compromised in their paracellular tightness, we found SARS-CoV-2 in the basolateral compartment in transwell assays after apical infection, suggesting active replication and transcellular transport of virus across the blood-brain barrier (BBB) in vitro. Moreover, entry of SARS-CoV-2 into BCECs could be reduced by anti-spike-, anti-angiotensin-converting enzyme 2 (ACE2)-, and anti-neuropilin-1 (NRP1)-specific antibodies or the transmembrane protease serine subtype 2 (TMPRSS2) inhibitor nafamostat. Together, our data provide strong support for SARS-CoV-2 brain entry across the BBB resulting in increased interferon signaling.

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, Salmonella infection, or pancreatic cancer. Autophagy activation versus inhibition strategies are being explored, while the role of autophagy in pathophysiology is being studied in parallel. However, the progress of preclinical and clinical development of autophagy modulators has been greatly hampered by the paucity of selective pharmacological agents and biomarkers to dissect their precise impact on various forms of autophagy and cellular responses. Here, we summarize established and new strategies in autophagy-related drug discovery and indicate a path toward establishing a more efficient discovery of autophagy-selective pharmacological agents. With this knowledge at hand, modern concepts for therapeutic exploitation of autophagy might become more plausible.Abbreviations: ALS: amyotrophic lateral sclerosis; AMPK: AMP-activated protein kinase; ATG: autophagy-related gene; AUTAC: autophagy-targeting chimera; CNS: central nervous system; CQ: chloroquine; GABARAP: gamma-aminobutyric acid type A receptor-associated protein; HCQ: hydroxychloroquine; LYTAC: lysosome targeting chimera; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NDD: neurodegenerative disease; PDAC: pancreatic ductal adenocarcinoma; PE: phosphatidylethanolamine; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; PROTAC: proteolysis-targeting chimera; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1.

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.

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 (1.5, 3, 5 µm). Against the intuitive expectation, a reduced proliferation was found on the arrays with the smallest array pitch of 1 µm and long NWs. Typically, cells settle in a fakir-like state on such densely-spaced nanowires and thus experience no substantial stress caused by nanowires indenting the cell membrane. However, imaging of F-actin showed a distinct reorganization of the cytoskeleton along the nanowire tips in the case of small array pitches interfering with regular proliferation. For larger pitches, the cell numbers depend on the NW lengths but proliferation generally continued although heavy deformations of the cell membrane were observed caused by the encapsulation of the nanowires. Moreover, we noticed a strong interaction of the nanowires with the nucleus in terms of squeezing and indenting. Remarkably, the cell viability is maintained at about 85% despite the massive deformation of the cells. Considering the enormous potential of human induced stem cells to study neurodegenerative diseases and the high cellular viability combined with a strong interaction with nanowire arrays, we believe that our results pave the way to apply nanowire arrays to human stem cells for future applications in stem cell research and regenerative medicine.

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. These should allow a preservation in their physiological, adherent state, an efficient re-cultivation and upscaling upon thawing towards high-throughput applications in cell therapies or disease modelling in drug discovery. Here, we present a novel vitrification-based method for adherent hiPSCs, designed for automated handling by microfluidic approaches and with ready-to-use potential e.g. in suspension-based bioreactors after thawing. Modifiable alginate microcarriers serve as a growth surface for adherent hiPSCs that were cultured in a suspension-based bioreactor and subsequently cryopreserved via droplet-based vitrification in comparison to conventional slow freezing. Soft (0.35%) versus stiff (0.65%) alginate microcarriers in concert with adhesion time variation have been examined. Findings revealed specific optimal conditions leading to an adhesion time and growth surface (matrix) elasticity dependent hypothesis on cryo-induced damaging regimes for adherent cell types. Deviations from the found optimum parameters give rise to membrane ruptures assessed via SEM and major cell loss after adherent vitrification. Applying the optimal conditions, droplet-based vitrification was superior to conventional slow freezing. A decreased microcarrier stiffness was found to outperform stiffer material regarding cell recovery, whereas the stemness characteristics of rewarmed hiPSCs were preserved.

Neuronal metabotropic glutamate receptor 8 protects against neurodegeneration in CNS inflammation.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with continuous neuronal loss. Treatment of clinical progression remains challenging due to lack of insights into inflammation-induced neurodegenerative pathways. Here, we show that an imbalance in the neuronal receptor interactome is driving glutamate excitotoxicity in neurons of MS patients and identify the MS risk-associated metabotropic glutamate receptor 8 (GRM8) as a decisive modulator. Mechanistically, GRM8 activation counteracted neuronal cAMP accumulation, thereby directly desensitizing the inositol 1,4,5-trisphosphate receptor (IP3R). This profoundly limited glutamate-induced calcium release from the endoplasmic reticulum and subsequent cell death. Notably, we found Grm8-deficient neurons to be more prone to glutamate excitotoxicity, whereas pharmacological activation of GRM8 augmented neuroprotection in mouse and human neurons as well as in a preclinical mouse model of MS. Thus, we demonstrate that GRM8 conveys neuronal resilience to CNS inflammation and is a promising neuroprotective target with broad therapeutic implications.

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). In mice, neuronal overexpression of Ppargc1a, which encodes for PGC-1α, led to increased numbers of mitochondria, complex IV activity, and maximum respiratory capacity. Moreover, Ppargc1a-overexpressing neurons showed a higher mitochondrial membrane potential that related to an improved calcium buffering capacity. Accordingly, neuronal deletion of Ppargc1a aggravated neurodegeneration during experimental autoimmune encephalomyelitis, while neuronal overexpression of Ppargc1a 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.

Multiple sclerosis is a life-long neurological condition that typically begins when people are in their twenties or thirties. Symptoms vary between individuals, and within a single individual over time, but can include difficulties with vision, balance, movement and thinking. These occur because the immune system of people with multiple sclerosis attacks the brain and spinal cord. This immune assault damages neurons and can eventually cause them to die. But exactly how this happens is unclear, and there are no drugs available that can prevent it. One idea is that the immune attack in multiple sclerosis damages neurons by disrupting structures inside them called mitochondria. These cellular 'organs', or organelles, produce the energy that all cells need to function correctly. If the mitochondria fail to generate enough energy, the cells can die. And because neurons are very active cells with high energy demands, they are particularly vulnerable to the effects of mitochondrial damage. By studying a mouse version of multiple sclerosis, Rosenkranz et al. now show that mitochondria in the neurons of affected animals are less active than those of healthy control mice. This is because the genes inside mitochondria that enable the organelles to produce energy are less active in the multiple sclerosis mice. Most of these genes that determine mitochondrial activity and energy production are under the control of a single master gene called PGC-1alpha. Rosenkranz et al. showed that boosting the activity of this gene ­ by introducing extra copies of it into neurons ­ increases mitochondrial activity in mice. It also makes the animals more resistant to the effects of multiple sclerosis. Boosting the activity of mitochondria in neurons could thus be a worthwhile therapeutic strategy to investigate for multiple sclerosis. Future studies should examine whether drugs that activate PGC-1alpha, for example, could help prevent neuronal death and the resulting symptoms of multiple sclerosis.

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. 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.

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. We here review the potential of mono- or (isogenic) co-culture BBB models based on brain capillary endothelial cells (BCECs) derived from human-induced pluripotent stem cells (hiPSCs), and compare them to several available BBB in vitro models from primary human or non-human cells and to rodent in vivo models, as well as to classical and widely used barrier models [Caco-2, parallel artificial membrane permeability assay (PAMPA)]. In particular, we are discussing the features and predictivity of these models and how hiPSC-derived BBB models could impact future discovery and development of novel CNS-targeting therapeutics. © 2020 The Authors.

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. 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.

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. While cell viability is maintained on all nanowire substrates, the settling regime of the cells can be controlled and tuned by the nanowire density from a fakir-like state to a complete nanowire wrapping state. Especially, full electrophysiological integrity of the neurons independent of the settling regime has been revealed by patch clamp experiments showing characteristic action potentials. Based on these results, our protocol has the potential to open new pathways in stem cell research and regenerative medicine utilizing human stem cell-derived neurons on tailor-made nanostructured substrates.