Cellular Pathomechanisms Research Articles

Abnormal functional lateralization and cooperation in bipolar disorder are associated with neurotransmitter and cellular profiles

BackgroundHemispheric lateralization and cooperation are essential for efficient brain function, and aberrations in both have been found in psychiatric disorders such as schizophrenia. This study investigated alterations in hemispheric lateralization and cooperation among patients with bipolar disorder (BD) and associations with neurotransmitter and cell-type density distributions to identify potential molecular and cellular pathomechanisms. MethodsSixty-seven BD patients and 127 healthy controls (HCs) were examined by resting-state functional MRI (rs-fMRI). Whole-brain maps of the autonomy index (AI) and connectivity between functionally homotopic voxels (CFH) were constructed to reveal BD-specific changes in brain functional lateralization and interhemispheric cooperation, respectively. Spatial associations of regional AI and CFH abnormalities with neurotransmitter and cell-type density distributions were examined by correlation analyses. ResultsBipolar disorder patients exhibited higher AI values in left superior parietal gyrus, cerebellar right Crus I, and cerebellar right Crus II, and these regional abnormalities were associated with the relative densities (proportions) of oligodendrocyte precursor cells and microglia. Patients also exhibited lower CFH values in right inferior parietal gyrus, bilateral middle occipital gyrus, left postcentral gyrus, and bilateral cerebellar crus II, and these regional abnormalities were associated with the densities of serotonin 1A and dopamine D2 receptors, oligodendrocyte precursor cells, astrocytes, and neurons. ConclusionsThese findings indicate that abnormal functional lateralization and cooperation in BD with potential molecular and cellular basis.

Loss of Fic causes progressive neurodegeneration in a Drosophila model of hereditary spastic paraplegia

Hereditary Spastic Paraplegia (HSP) is a group of rare inherited disorders characterized by progressive weakness and spasticity of the legs. Recent newly discovered biallelic variants in the gene FICD were found in patients with a highly similar phenotype to early onset HSP. FICD encodes filamentation induced by cAMP domain protein. FICD is involved in the AMPylation and deAMPylation protein modifications of the endoplasmic reticulum (ER) chaperone BIP, a major constituent of the ER that regulates the unfolded protein response. Although several biochemical properties of FICD have been characterized, the neurological function of FICD and the pathological mechanism underlying HSP are unknown. We established a Drosophila model to gain mechanistic understanding of the function of FICD in HSP pathogenesis, and specifically the role of BIP in neuromuscular physiology. Our studies on Drosophila Fic null mutants uncovered that loss of Fic resulted in locomotor impairment and reduced levels of BIP in the motor neuron circuitry, as well as increased reactive oxygen species (ROS) in the ventral nerve cord of Fic null mutants. Finally, feeding Drosophila Fic null mutants with chemical chaperones PBA or TUDCA, or treatment of patient fibroblasts with PBA, reduced the ROS accumulation. The neuronal phenotypes of Fic null mutants recapitulate several clinical features of HSP patients and further reveal cellular patho-mechanisms. By modeling FICD in Drosophila, we provide potential targets for intervention for HSP, and advance fundamental biology that is important for understanding related rare and common neuromuscular diseases.

Energy metabolism disturbance, altered neuronal development and glutamatergic signalling in human derived neuronal cell models of ADHD

Abstract Despite major advances in research into the neurobiological basis of mental illness, there have been hardly any new developments in new drug therapies. As there are approximately 30% of affected individuals that do not respond sufficiently to available treatments, there is a significant unmet medical need for new therapeutic approaches. About 90% of novel substances that have shown promise in animal studies are not effective in clinical trials. Recent research on human induced pluripotent stem cells (hiPSC) could lead to the use of more human-tailored models in this field. IPSC-derived cell models and organoids may be very attractive for preclinical screening and bridge the gap between in vitro and in vivo studies, reducing animal testing. However, the next steps must first demonstrate the validity and reproducibility of the initial functional results from the hIPSC models of mental illness. In our own studies on neuronal cell models of patients with attention-deficit/hyperactivity disorder (ADHD) with rare PARK2 gene variants, we were able to show evidence of mitochondrial dysfunction and impaired energy metabolism. Additionally, we have first hints at a oxidative dysbalance which could be as well targeted by medication. In a model of cortical development of ADHD patients with common variants in the ADGRL3 gene, we found first evidence for altered neuronal maturation as well as abnormalities in calcium metabolism and glutamatergic functionality compared to cells from healthy controls. In summary, these first results are promising that hIPSC models can contribute new insights into cellular pathomechanisms of mental and neurodevelopmental disorders and the development of new, individualised therapeutic approaches.Disclosure of InterestS. Kittel-Schneider Grant / Research support from: The studies are funded by IZKF Wuerzburg and BBRF fund (to SKS and RMcN). SKS received speaker’s honoraria from Takeda, Medice and Janssen.

Loss of function of FIP200 in human pluripotent stem cell-derived neurons leads to axonal pathology and hyperactivity

FIP200 plays important roles in homeostatic processes such as autophagy and signaling pathways such as focal adhesion kinase (FAK) signaling. Furthermore, genetic studies suggest an association of FIP200 mutations with psychiatric disorders. However, its potential connections to psychiatric disorders and specific roles in human neurons are not clear. We set out to establish a human-specific model to study the functional consequences of neuronal FIP200 deficiency. To this end, we generated two independent sets of isogenic human pluripotent stem cell lines with homozygous FIP200KO alleles, which were then used for the derivation of glutamatergic neurons via forced expression of NGN2. FIP200KO neurons exhibited pathological axonal swellings, showed autophagy deficiency, and subsequently elevated p62 protein levels. Moreover, monitoring the electrophysiological activity of neuronal cultures on multi-electrode arrays revealed that FIP200KO resulted in a hyperactive network. This hyperactivity could be abolished by glutamatergic receptor antagonist CNQX, suggesting a strengthened glutamatergic synaptic activation in FIP200KO neurons. Furthermore, cell surface proteomic analysis revealed metabolic dysregulation and abnormal cell adhesion-related processes in FIP200KO neurons. Interestingly, an ULK1/2-specific autophagy inhibitor could recapitulate axonal swellings and hyperactivity in wild-type neurons, whereas inhibition of FAK signaling was able to normalize the hyperactivity of FIP200KO neurons. These results suggest that impaired autophagy and presumably also disinhibition of FAK can contribute to the hyperactivity of FIP200KO neuronal networks, whereas pathological axonal swellings are primarily due to autophagy deficiency. Taken together, our study reveals the consequences of FIP200 deficiency in induced human glutamatergic neurons, which might, in the end, help to understand cellular pathomechanisms contributing to neuropsychiatric conditions.

Ferroptosis Inhibitors as New Therapeutic Insights into Radiation-Induced Heart Disease.

Radiation-induced heart disease (RIHD) is a significant cause of morbidity in breast and other mediastinal cancers. The many molecular and cellular patho-mechanisms that have a role in RIHD are not completely understood. Endothelial injury, oxidative stress, and inflammation, as well as endoplasmic reticulum and mitochondrial damage, are considered the primary causes of RIHD. Ferroptosis is a newly discovered type of cell death that results from irondependent lipid peroxide accumulation. As ferroptosis plays an important role in the pathogenesis of cardiovascular diseases, it seems that it has a significant effect on RIHD. It was recently shown that ionizing radiation (IR) generates severe ferroptosis, which is a critical component of Radiotherapy-mediated normal cell toxicity. These findings support the use of a ferroptosis inhibitor to reduce RIHD. In this perspective review, we summarize the role of ferroptosis in pathogens of cardiovascular disease and radiation toxicity, and we will introduce ferroptosis inhibitors as a new strategy to prevent or reduce RIHD.

Loss of surface transport is a main cellular pathomechanism of CRB2 variants causing podocytopathies.

Crumbs2 (CRB2) is a central component of the renal filtration barrier and part of the slit diaphragm, a unique cell contact formed by glomerular podocytes. Some CRB2 variants cause recessive inherited forms of steroid-resistant nephrotic syndrome. However, the disease-causing potential of numerous CRB2 variants remains unknown. Here, we report the establishment of a live-cell imaging-based assay, allowing a quantitative evaluation of the pathogenic potential of so far non-categorized CRB2 variants. Based on in silico data analysis and protein prediction software, putative disease-associated CRB2 missense variants were selected, expressed as CRB2-GFP fusion proteins, and analyzed in reporter cell lines with BFP-labeled plasma membrane. We found that in comparison with PM-localized WT, disease-associated CRB2 variants remained predominantly at the ER. Accumulation at the ER was also present for several non-characterized CRB2 variants and variants in which putative disulfide bridge-forming cysteines were replaced. Strikingly, WT CRB2 retained inside the ER in cells lacking protein disulfide isomerase A3, indicating that posttranslational modification, especially the formation of disulfide bridges, is a crucial step for the CRB2 PM transport.

A Comparative Presentation of Mouse Models That Recapitulate Most Features of Alport Syndrome.

Alport syndrome is a hereditary kidney disease caused by mutations in the three genes encoding for collagen IV: COL4A3, COL4A4, and COL4A5. Several mouse models have been created for the study of this disease with variable phenotypic outcomes. This review is an up-to-date presentation of the current mouse models existing in the literature with a detailed comparison of the phenotypic features characterizing each model. Although in humans it is primarily a glomerulopathy, data suggest that in some mouse models, the initial symptoms appear in the tubule-interstitial region rather than the glomerulus. Additionally, in some other models, the severity of disease in the tubule-interstitial region is affected by the genetic background. In conclusion, the phenotypic spectrum of each model appears to be affected by the model’s genetic background, the position of the genetic alteration within the gene, and the type of the genetic alteration. Despite these disparities, mouse models recapitulate with relatively high fidelity several features of the human disease, which makes them useful for studies aimed at better understanding cellular pathomechanisms and for finding new treatments.

Generation and validation of an iPSC line (BBANTWi008-A) from a Loeys-Dietz Syndrome type 3 patient

Loeys-Dietz Syndrome (LDS) is an autosomal dominant connective tissue disorder. The major hallmark of LDS is thoracic aortic aneurysm and dissection (TAAD). We generated an induced pluripotent stem cell (iPSC) line of a severely affected LDS patient carrying a pathogenic SMAD3 p.Arg287Gln variant. Peripheral blood mononuclear cells were reprogrammed using non-integrating Sendai viral vectors. The autonomous pluripotency state of the resulting iPSC model was proven by the presence of pluripotency markers, trilineage differentiation potential and absence of the Sendai vector backbone. This iPSC line can be used to study and/or therapeutically target the cellular pathomechanisms of SMAD3-related LDS.

Structural Analysis of Mitochondrial Dynamics-From Cardiomyocytes to Osteoblasts: A Critical Review.

Mitochondria play a crucial role in cell physiology and pathophysiology. In this context, mitochondrial dynamics and, subsequently, mitochondrial ultrastructure have increasingly become hot topics in modern research, with a focus on mitochondrial fission and fusion. Thus, the dynamics of mitochondria in several diseases have been intensively investigated, especially with a view to developing new promising treatment options. However, the majority of recent studies are performed in highly energy-dependent tissues, such as cardiac, hepatic, and neuronal tissues. In contrast, publications on mitochondrial dynamics from the orthopedic or trauma fields are quite rare, even if there are common cellular mechanisms in cardiovascular and bone tissue, especially regarding bone infection. The present report summarizes the spectrum of mitochondrial alterations in the cardiovascular system and compares it to the state of knowledge in the musculoskeletal system. The present paper summarizes recent knowledge regarding mitochondrial dynamics and gives a short, but not exhaustive, overview of its regulation via fission and fusion. Furthermore, the article highlights hypoxia and its accompanying increased mitochondrial fission as a possible link between cardiac ischemia and inflammatory diseases of the bone, such as osteomyelitis. This opens new innovative perspectives not only for the understanding of cellular pathomechanisms in osteomyelitis but also for potential new treatment options.

Primary Cilia Structure Is Prolonged in Enteric Neurons of 5xFAD Alzheimer's Disease Model Mice.

Neurodegenerative diseases such as Alzheimer’s disease (AD) have long been acknowledged as mere disorders of the central nervous system (CNS). However, in recent years the gut with its autonomous nervous system and the multitude of microbial commensals has come into focus. Changes in gut properties have been described in patients and animal disease models such as altered enzyme secretion or architecture of the enteric nervous system. The underlying cellular mechanisms have so far only been poorly investigated. An important organelle for integrating potentially toxic signals such as the AD characteristic A-beta peptide is the primary cilium. This microtubule-based signaling organelle regulates numerous cellular processes. Even though the role of primary cilia in a variety of developmental and disease processes has recently been recognized, the contribution of defective ciliary signaling to neurodegenerative diseases such as AD, however, has not been investigated in detail so far. The AD mouse model 5xFAD was used to analyze possible changes in gut functionality by organ bath measurement of peristalsis movement. Subsequently, we cultured primary enteric neurons from mutant mice and wild type littermate controls and assessed for cellular pathomechanisms. Neurite mass was quantified within transwell culturing experiments. Using a combination of different markers for the primary cilium, cilia number and length were determined using fluorescence microscopy. 5xFAD mice showed altered gut anatomy, motility, and neurite mass of enteric neurons. Moreover, primary cilia could be demonstrated on the surface of enteric neurons and exhibited an elongated phenotype in 5xFAD mice. In parallel, we observed reduced β-Catenin expression, a key signaling molecule that regulates Wnt signaling, which is regulated in part via ciliary associated mechanisms. Both results could be recapitulated via in vitro treatments of enteric neurons from wild type mice with A-beta. So far, only a few reports on the probable role of primary cilia in AD can be found. Here, we reveal for the first time an architectural altered phenotype of primary cilia in the enteric nervous system of AD model mice, elicited potentially by neurotoxic A-beta. Potential changes on the sub-organelle level—also in CNS-derived neurons—require further investigations.

Cardiac muscle disease and therapeutic targets

Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. While ∼50% of patients with HCM carry a sarcomere gene mutation (sarcomere mutation-positive, SMP), the genetic background is unknown in the other half of the patients (sarcomere mutation-negative, SMN). Gene mutations are most often present in genes encoding the sarcomere proteins myosin heavy chain, myosin-binding protein C, and troponin T. Studies in cardiac tissue samples from patients with obstructive HCM that were obtained during myectomy surgery showed increased myofilament calcium sensitivity, increased kinetics and tension cost, and a reduction of the super-relaxed state of myosin, which is associated with an energy-conserving status of the crossbridges. The increase in myofilament calcium sensitivity is observed at a low dose of mutant protein, while the magnitude of the increase in calcium sensitivity depends on the specific mutation location. These mutation-mediated myofilament changes may underlie inefficient in vivo cardiac performance in mutation carriers. Reduced cardiac efficiency has been observed before onset of cardiac hypertrophy and at advanced disease stages. In addition, impaired diastolic function is an early disease characteristic of HCM. Our recent proteomics studies revealed increased detyrosination of microtubules, which may be a cause of diastolic dysfunction. Recent treatments that target inefficient cardiac performance, such as myosin inhibitors and metabolic drug therapies, may have the potential to prevent, delay, or even reverse disease in HCM-mutation carriers. Treatment response may depend on the specific gene mutation in SMP individuals and may explain diverse response of HCM patients to therapy. While mutation-mediated cardiomyocyte defects have become clear in past years, more research is warranted to define the cellular pathomechanisms of cardiac dysfunction in SMN patients.

Modelling Mitochondrial Disease in Human Pluripotent Stem Cells: What Have We Learned?

Mitochondrial diseases disrupt cellular energy production and are among the most complex group of inherited genetic disorders. Affecting approximately 1 in 5000 live births, they are both clinically and genetically heterogeneous, and can be highly tissue specific, but most often affect cell types with high energy demands in the brain, heart, and kidneys. There are currently no clinically validated treatment options available, despite several agents showing therapeutic promise. However, modelling these disorders is challenging as many non-human models of mitochondrial disease do not completely recapitulate human phenotypes for known disease genes. Additionally, access to disease-relevant cell or tissue types from patients is often limited. To overcome these difficulties, many groups have turned to human pluripotent stem cells (hPSCs) to model mitochondrial disease for both nuclear-DNA (nDNA) and mitochondrial-DNA (mtDNA) contexts. Leveraging the capacity of hPSCs to differentiate into clinically relevant cell types, these models permit both detailed investigation of cellular pathomechanisms and validation of promising treatment options. Here we catalogue hPSC models of mitochondrial disease that have been generated to date, summarise approaches and key outcomes of phenotypic profiling using these models, and discuss key criteria to guide future investigations using hPSC models of mitochondrial disease.

Regional microglial populations in central autonomic brain regions in SUDEP

Sudden unexpected death in epilepsy (SUDEP) may arise as a result of autonomic dysfunction during a seizure. The central autonomic networks (CANs) modulate brainstem cardiorespiratory regulation. Recent magnetic resonance imaging (MRI) studies in SUDEP have shown cortical and subcortical volume changes and altered connectivity between CAN regions, but the pathological correlate is unknown. Because neuroinflammation is both a cause and a consequence of seizures and may relate to regional brain pathology, our aim was to evaluate microglial populations in CANs in SUDEP. In 55 postmortem cases, including SUDEP, epilepsy controls without SUDEP and nonepilepsy controls, we quantified Iba1-expressing microglia in 14 cortical and thalamic areas that included known CAN regions. Mean Iba1 labeling across all brain regions was significantly higher in SUDEP cases compared to epilepsy and nonepilepsy controls. There was significant regional variation in Iba1 labeling in SUDEP cases only, with highest labeling in the medial thalamus. Significantly higher labeling in SUDEP cases than epilepsy and nonepilepsy controls was consistently noted in the superior temporal gyrus. In cases with documented seizures up to 10 days prior to death, significantly higher mean Iba1 labeling was observed in SUDEP compared to epilepsy controls. Our findings support microglial activation in SUDEP, including cortical and subcortical regions with known autonomic functions such as the thalamus and superior temporal gyrus. This may be relevant to cellular pathomechanisms underlying cardioregulatory failure during a seizure.

New insights into the clinical and molecular spectrum of the novel CYFIP2-related neurodevelopmental disorder and impairment of the WRC-mediated actin dynamics

PurposeA few de novo missense variants in the cytoplasmic FMRP-interacting protein 2 (CYFIP2) gene have recently been described as a novel cause of severe intellectual disability, seizures, and hypotonia in 18 individuals, with p.Arg87 substitutions in the majority.MethodsWe assembled data from 19 newly identified and all 18 previously published individuals with CYFIP2 variants. By structural modeling and investigation of WAVE-regulatory complex (WRC)-mediated actin polymerization in six patient fibroblast lines we assessed the impact of CYFIP2 variants on the WRC.ResultsSixteen of 19 individuals harbor two previously described and 11 novel (likely) disease-associated missense variants. We report p.Asp724 as second mutational hotspot (4/19 cases). Genotype–phenotype correlation confirms a consistently severe phenotype in p.Arg87 patients but a more variable phenotype in p.Asp724 and other substitutions. Three individuals with milder phenotypes carry putative loss-of-function variants, which remain of unclear pathogenicity. Structural modeling predicted missense variants to disturb interactions within the WRC or impair CYFIP2 stability. Consistent with its role in WRC-mediated actin polymerization we substantiate aberrant regulation of the actin cytoskeleton in patient fibroblasts.ConclusionOur study expands the clinical and molecular spectrum of CYFIP2-related neurodevelopmental disorder and provides evidence for aberrant WRC-mediated actin dynamics as contributing cellular pathomechanism.

A homozygous DSC2 deletion associated with arrhythmogenic cardiomyopathy is caused by uniparental isodisomy

AimsWe aimed to unravel the genetic, molecular and cellular pathomechanisms of DSC2 truncation variants leading to arrhythmogenic cardiomyopathy (ACM). Methods and resultsWe report a homozygous 4-bp DSC2 deletion variant c.1913_1916delAGAA, p.Q638LfsX647hom causing a frameshift carried by an ACM patient. Whole exome sequencing and comparative genomic hybridization analysis support a loss of heterozygosity in a large segment of chromosome 18 indicating segmental interstitial uniparental isodisomy (UPD). Ultrastructural analysis of the explanted myocardium from a mutation carrier using transmission electron microscopy revealed a partially widening of the intercalated disc. Using qRT-PCR we demonstrated that DSC2 mRNA expression was substantially decreased in the explanted myocardial tissue of the homozygous carrier compared to controls. Western blot analysis revealed absence of both full-length desmocollin-2 isoforms. Only a weak expression of the truncated form of desmocollin-2 was detectable. Immunohistochemistry showed that the truncated form of desmocollin-2 did not localize at the intercalated discs. In vitro, transfection experiments using induced pluripotent stem cell derived cardiomyocytes and HT-1080 cells demonstrated an obvious absence of the mutant truncated desmocollin-2 at the plasma membrane. Immunoprecipitation in combination with fluorescence measurements and Western blot analyses revealed an abnormal secretion of the truncated desmocollin-2. ConclusionIn summary, we unraveled segmental UPD as the likely genetic reason for a small homozygous DSC2 deletion. We conclude that a combination of nonsense mediated mRNA decay and extracellular secretion is involved in DSC2 related ACM.

Fluorinated Galactoses Inhibit Galactose-1-Phosphate Uridyltransferase and Metabolically Induce Galactosemia-like Phenotypes in HEK-293 Cells.

Genetic defects of human galactose-1-phosphate uridyltransferase (hGALT) and the partial loss of enzyme function result in an altered galactose metabolism with serious long-term developmental impairment of organs in classic galactosemia patients. In search for cellular pathomechanisms induced by the stressor galactose, we looked for ways to induce metabolically a galactosemia-like phenotype by hGALT inhibition in HEK293 cells. In kinetic studies, we provide evidence for 2-fluorinated galactose-1-phosphate (F-Gal-1-P) to competitively inhibit recombinant hGALT with a KI of 0.9 mM. Contrasting with hepatic cells, no alterations of N-glycoprofiles in MIG (metabolic induction of galactosemia)-HEK293 cells were revealed for an inducible secretory netrin-1 probe by MALDI-MS. Differential fluorescence-activated cell sorting demonstrated reduced surface expression of N-glycosylated CD109, EGFR, DPP4, and rhMUC1. Membrane raft proteomes exhibited dramatic alterations pointing to an affection of the unfolded protein response, and of targeted protein traffick. Most prominent, a negative regulation of oxidative stress was revealed presumably as a response to a NADPH pool depletion during reduction of Gal/F-Gal. Cellular perturbations induced by fluorinated galactoses in normal epithelial cells resemble proteomic changes revealed for galactosemic fibroblasts. In conclusion, the metabolic induction of galactosemia-like phenotypes in healthy epithelial/neuronal cells could support studies on the molecular pathomechanisms in classic galactosemia, in particular under conditions of low galactose stress and residual GALT activity.

Astroglia in Leukodystrophies.

Leukodystrophies are genetically determined disorders affecting the white matter of the central nervous system. The combination of MRI pattern recognition and next-generation sequencing for the definition of novel disease entities has recently demonstrated that many leukodystrophies are due to the primary involvement and/or mutations in genes selectively expressed by cell types other than the oligodendrocytes, the myelin-forming cells in the brain. This has led to a new definition of leukodystrophies as genetic white matter disorders resulting from the involvement of any white matter structural component. As a result, the research has shifted its main focus from oligodendrocytes to other types of neuroglia. Astrocytes are the housekeeping cells of the nervous system, responsible for maintaining homeostasis and normal brain physiology and to orchestrate repair upon injury. Several lines of evidence show that astrocytic interactions with the other white matter cellular constituents play a primary pathophysiologic role in many leukodystrophies. These are thus now classified as astrocytopathies. This chapter addresses how the crosstalk between astrocytes, other glial cells, axons and non-neural cells are essential for the integrity and maintenance of the white matter in health. It also addresses the current knowledge of the cellular pathomechanisms of astrocytic leukodystrophies, and specifically Alexander disease, vanishing white matter, megalencephalic leukoencephalopathy with subcortical cysts and Aicardi-Goutière Syndrome.

Yeast as a model to study protein misfolding in aged cells.

Yeast models of neurodegenerative diseases associated with protein misfolding and protein aggregation have given unique insights into the underlying genetic and cellular pathomechanisms. These yeast models recapitulate central aspects of protein misfolding and the ensuing toxicity, such as interference with cellular protein quality control, concentration-dependent formation of insoluble, often amyloid-like aggregates and the associated toxicity. Advanced age is undoubtedly the highest and most common risk factor for most neurodegenerative diseases. Since yeast has served as a superb model to study cellular aspects of aging, we outline strategies to study how aging modulates protein misfolding and its toxicity, thereby opening new avenues to continue the success of yeast as powerful models to study neurodegenerative diseases.

Cell Type-Specific Mechanisms in the Pathogenesis of Ischemic Stroke: The Role of Apoptosis Signal-Regulating Kinase 1.

Stroke has become a more common disease worldwide. Despite great efforts to develop treatment, little is known about ischemic stroke. Cerebral ischemia activates multiple cascades of cell type-specific pathomechanisms. Ischemic brain injury consists of a complex series of cellular reactions in various cell types within the central nervous system (CNS) including platelets, endothelial cells, astrocytes, neutrophils, microglia/macrophages, and neurons. Diverse cellular changes after ischemic injury are likely to induce cell death and tissue damage in the brain. Since cells in the brain exhibit different functional roles at distinct time points after injury (acute/subacute/chronic phases), it is difficult to pinpoint genuine roles of cell types after brain injury. Many experimental studies have shown the association of apoptosis signal-regulating kinase 1 (ASK1) with cellular pathomechanisms after cerebral ischemia. Blockade of ASK1, by either pharmacological or genetic manipulation, leads to reduced ischemic brain injury and subsequent neuroprotective effects. In this review, we present the cell type-specific pathophysiology of the early phase of ischemic stroke, the role of ASK1 suggested by preclinical studies, and the potential use of ASK suppression, either by pharmacologic or genetic suppression, as a promising therapeutic option for ischemic stroke recovery.

Cardiomyocyte Hypocontractility and Reduced Myofibril Density in End-Stage Pediatric Cardiomyopathy.

Dilated cardiomyopathy amongst children (pediatric cardiomyopathy, pediatric CM) is associated with a high morbidity and mortality. Because little is known about the pathophysiology of pediatric CM, treatment is largely based on adult heart failure therapy. The reason for high morbidity and mortality is largely unknown as well as data on cellular pathomechanisms is limited. Here, we assessed cardiomyocyte contractility and protein expression to define cellular pathomechanisms in pediatric CM. Explanted heart tissue of 11 pediatric CM patients and 18 controls was studied. Contractility was measured in single membrane-permeabilized cardiomyocytes and protein expression was assessed with gel electrophoresis and western blot analysis. We observed increased Ca2+-sensitivity of myofilaments which was due to hypophosphorylation of cardiac troponin I, a feature commonly observed in adult DCM. We also found a significantly reduced maximal force generating capacity of pediatric CM cardiomyocytes, as well as a reduced passive force development over a range of sarcomere lengths. Myofibril density was reduced in pediatric CM compared to controls. Correction of maximal force and passive force for myofibril density normalized forces in pediatric CM cardiomyocytes to control values. This implies that the hypocontractility was caused by the reduction in myofibril density. Unlike in adult DCM we did not find an increase in compliant titin isoform expression in end-stage pediatric CM. The limited ability of pediatric CM patients to maintain myofibril density might have contributed to their early disease onset and severity.