Neural Cell Model Research Articles

Protective Mechanism of Proprotein Convertase Subtilisin-Like Kexin Type 9 Inhibitor on Rats with Middle Cerebral Artery Occlusion-Induced Cerebral Ischemic Infarction.

The aim of this study was to research the mechanism of proprotein convertase subtilisin-like kexin type 9 (PCSK9) inhibitor in neural protective effect on rat cerebral ischemic reperfusion injury (I/RI). The transient middle cerebral artery occlusion (tMCAO) model of rats was prepared by the suture method, and PCSK9 inhibitor was injected intraperitoneally immediately after I/R. The rats were scored for neurological deficits and the cerebral infarction volume was measured. The brain tissues were collected and western blot (WB) was used to detect the expression of PCSK9. The rat cortical neural stem cells were treated with oxygen glucose deprivation (OGD) to establish a cell model of ischemia/reperfusion. WB was used to detect the expression of PCSK9 and the apoptosis-related pathway proteins. After interfering with the expression of PCSK9 siRNA, the cell viability (cell counting kit-8 assay) and apoptosis (TUNEL staining, Annexin V/PI method) were detected, and the cell proliferation was detected by EdU staining and flow cytometry. The expression of PCSK9 in the brain tissue of the MCAO group was dramatically increased. PCSK9 inhibitor can improve neurobehavioral scores and reduce apoptosis and infarct volume. An OGD model of neural stem cells in vitro was constructed. Inhibiting PCSK9 with si-PCSK9 can increase cell viability, promote cell proliferation, and also reduce cell apoptosis. Inhibition of PCSK9 can decrease the cerebral infarct volume in rats with cerebral I/RI and improve the neural function. Mechanically, inhibition of PCSK9 can lead to the decrease of nerve cell apoptosis and promotion of cell proliferation.

Glutamate Uptake Is Not Impaired by Hypoxia in a Culture Model of Human Fetal Neural Stem Cell-Derived Astrocytes.

Hypoxic ischemic injury to the fetal and neonatal brain is a leading cause of death and disability worldwide. Although animal and culture studies suggest that glutamate excitotoxicity is a primary contributor to neuronal death following hypoxia, the molecular mechanisms, and roles of various neural cells in the development of glutamate excitotoxicity in humans, is not fully understood. In this study, we developed a culture model of human fetal neural stem cell (FNSC)-derived astrocytes and examined their glutamate uptake in response to hypoxia. We isolated, established, and characterized cultures of FNSCs from aborted fetal brains and differentiated them into astrocytes, characterized by increased expression of the astrocyte markers glial fibrillary acidic protein (GFAP), excitatory amino acid transporter 1 (EAAT1) and EAAT2, and decreased expression of neural stem cell marker Nestin. Differentiated astrocytes were exposed to various oxygen concentrations mimicking normoxia (20% and 6%), moderate and severe hypoxia (2% and 0.2%, respectively). Interestingly, no change was observed in the expression of the glutamate transporter EAAT2 or glutamate uptake by astrocytes, even after exposure to severe hypoxia for 48 h. These results together suggest that human FNSC-derived astrocytes can maintain glutamate uptake after hypoxic injury and thus provide evidence for the possible neuroprotective role of astrocytes in hypoxic conditions.

Impact of Organelle Transport Deficits on Mitophagy and Autophagy in Niemann-Pick Disease Type C.

Defective mitochondria are pathophysiological features of a number of neurodegenerative diseases. Here, we investigated mitochondrial dysfunction in the context of the rare lysosomal storage diseases Niemann–Pick disease type C1 and type C2 (NP-C1 and NP-C2). Mutations in either the NPC1 or NPC2 gene lead to cholesterol accumulation in late endosomes and lysosomes, resulting in impaired cholesterol homeostasis. The extent to which this may lead to mitochondrial dysfunction has been poorly studied so far. Therefore, we investigated the morphology, function, and transport of mitochondria, as well as their degradation via mitophagy, in a disease-associated human neural cell model of NP-C. By performing live cell imaging, we observed markedly reduced mitochondrial transport, although morphology and function were not appreciably altered. However, we observed a defective mitophagy induction shown by a reduced capability to elevate parkin expression and engulf mitochondria in autophagosomes after treatment with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). This was accompanied by defects in autophagy induction, exhibited by a hampered p62 expression and progression, shown by increased LC3BII levels and a defective fusion of autophagosomes and lysosomes. The latter might have been additionally influenced by the observed reduced lysosomal transport. Hence, we hypothesized that a reduced recycling of mitochondria contributes to the pathophysiology of NP-C.

Downregulated Calcium-Binding Protein S100A16 and HSP27 in Placenta-Derived Multipotent Cells Induce Functional Astrocyte Differentiation

Little is known about genes that induce stem cells differentiation into astrocytes. We previously described that heat shock protein 27 (HSP27) downregulation is directly related to neural differentiation under chemical induction in placenta-derived multipotent stem cells (PDMCs). Using this neural differentiation cell model, we cross-compared transcriptomic and proteomic data and selected 26 candidate genes with the same expression trends in both omics analyses. Those genes were further compared with a transcriptomic database derived from Alzheimer’s disease (AD). Eighteen out of 26 candidates showed opposite expression trends between our data and the AD database. The mRNA and protein expression levels of those candidates showed downregulation of HSP27, S100 calcium-binding protein A16 (S100A16) and two other genes in our neural differentiation cell model. Silencing these four genes with various combinations showed that co-silencing HSP27 and S100A16 has stronger effects than other combinations for astrocyte differentiation. The induced astrocyte showed typical astrocytic star-shape and developed with ramified, stringy and filamentous processes as well as differentiated endfoot structures. Also, some of them connected with each other and formed continuous network. Immunofluorescence quantification of various neural markers indicated that HSP27 and S100A16 downregulation mainly drive PDMCs differentiation into astrocytes. Immunofluorescence and confocal microscopic images showed the classical star-like shape morphology and co-expression of crucial astrocyte markers in induced astrocytes, while electrophysiology and Ca2+ influx examination further confirmed their functional characteristics. In conclusion, co-silencing of S100A16 and HSP27 without chemical induction leads to PDMCs differentiation into functional astrocytes.Graphical abstract

Herpesvirus Infections in the Human Brain: A Neural Cell Model of the Complement System Derived from Induced Pluripotent Stem Cells.

Herpesviruses alter cognitive functions in humans following acute infections; progressive cognitive decline and dementia have also been suggested. It is important to understand the pathogenic mechanisms of such infections. The complement system - comprising functionally related proteins integral for systemic innate and adaptive immunity - is an important component of host responses. The complement system has specialized functions in the brain. Still, the dynamics of the brain complement system are still poorly understood. Many complement proteins have limited access to the brain from plasma, necessitating synthesis and specific regulation of expression in the brain; thus, complement protein synthesis, activation, regulation, and signaling should be investigated in human brain-relevant cellular models. Cells derived from human-induced pluripotent stem cells (hiPSCs) could enable tractable models. Human-induced pluripotent stem cells were differentiated into neuronal (hi-N) and microglial (hi-M) cells that were cultured with primary culture human astrocyte-like cells (ha-D). Gene expression analyses and complement protein levels were analyzed in mono- and co-cultures. Transcript levels of complement proteins differ by cell type and co-culture conditions, with evidence for cellular crosstalk in co-cultures. Hi-N and hi-M cells have distinct patterns of expression of complement receptors, soluble factors, and regulatory proteins. hi-N cells produce complement factor 4 (C4) and factor B (FB), whereas hi-M cells produce complement factor 2 (C2) and complement factor 3 (C3). Thus, neither hi-N nor hi-M cells can form either of the C3-convertases - C4bC2a and C3bBb. However, when hi-N and hi-M cells are combined in co-cultures, both types of functional C3 convertase are produced, indicated by elevated levels of the cleaved C3 protein, C3a. hiPSC-derived co-culture models can be used to study viral infection in the brain, particularly complement receptor and function in relation to cellular "crosstalk." The models could be refined to further investigate pathogenic mechanisms.

SmartFlareTM is a reliable method for assessing mRNA expression in single neural stem cells.

BACKGROUNDOne of the most challenging tasks of modern biology concerns the real-time tracking and quantification of mRNA expression in living cells. On this matter, a novel platform called SmartFlareTM has taken advantage of fluorophore-linked nanoconstructs for targeting RNA transcripts. Although fluorescence emission does not account for the spatial mRNA distribution, NanoFlare technology has grown a range of theranostic applications starting from detecting biomarkers related to diseases, such as cancer, neurodegenerative pathologies or embryonic developmental disorders.AIMTo investigate the potential of SmartFlareTM in determining time-dependent mRNA expression of prominin 1 (CD133) and octamer-binding transcription factor 4 (OCT4) in single living cells through differentiation.METHODSBrain fragments from the striatum of aborted human fetuses aged 8 wk postconception were processed to obtain neurospheres. For the in vitro differentiation, neurospheres were gently dissociated with Accutase solution. Single cells were resuspended in a basic medium enriched with fetal bovine serum, plated on poly-L-lysine-coated glass coverslips, and grown in a lapse of time from 1 to 4 wk. Live cell mRNA detection was performed using SmartFlareTM probes (CD133, Oct4, Actin, and Scramble). All the samples were incubated at 37 °C for 24 h. For nuclear staining, Hoechst 33342 was added. SmartFlareTM CD133- and OCT4-specific fluorescence signal was assessed using a semiquantitative visual approach, taking into account the fluorescence intensity and the number of labeled cells.RESULTSIn agreement with previous PCR experiments, a unique expression trend was observed for CD133 and OCT4 genes until 7 d in vitro (DIV). Fluorescence resulted in a mixture of diffuse cytoplasmic and spotted-like pattern, also detectable in the contacting neural branches. From 15 to 30 DIV, only few cells showed a scattered fluorescent pattern, in line with the differentiation progression and coherent with mRNA downregulation of these stemness-related genes.CONCLUSIONSmartFlareTM appears to be a reliable, easy-to-handle tool for investigating CD133 and OCT4 expression in a neural stem cell model, preserving cell biological properties in anticipation of downstream experiments.

Micromechanical characterisation of 3D bioprinted neural cell models using Brillouin microspectroscopy

Biofabrication of three-dimensional (3D) in vitro neural cell models that closely mimic the central nervous system (CNS) is an emerging field of research with applications from fundamental biology to regenerative medicine, and far reaching benefits for the economy, healthcare and the ethical use of animals. The micromechanical properties of such models are an important factor dictating the success of modelling outcomes in relation to accurate reproduction of the processes in native tissues. Characterising the micromechanical properties of such models non-destructively and over a prolonged span of time, however, are key challenges. Brillouin microspectroscopy (BM) could provide a solution to this problem since this technology is non-invasive, label-free and is capable of micro-scale 3D imaging. In this work, the micromechanical properties of 3D bioprinted neural cell models consisting of NG 108-15 neuronal cells and Gelatin methacryloyl (GelMA) hydrogels of various concentrations were investigated using BM. We demonstrate changes in the volume-averaged (VA) and local micro-scale mechanical properties of these models over a 7 day period, in which the hydrogel component of the model are found to soften as the cells grow, multiply and form stiffer spheroid-type structures. These findings signify the necessity to resolve in microscopic detail the mechanics of in vitro 3D tissue models and suggest Brillouin microspectroscopy to be a suitable technology to bridge this gap.

Neurosurgical Approaches to Brain Tissue Harvesting for the Establishment of Cell Cultures in Neural Experimental Cell Models.

In recent decades, cell biology has made rapid progress. Cell isolation and cultivation techniques, supported by modern laboratory procedures and experimental capabilities, provide a wide range of opportunities for in vitro research to study physiological and pathophysiological processes in health and disease. They can also be used very efficiently for the analysis of biomaterials. Before a new biomaterial is ready for implantation into tissues and widespread use in clinical practice, it must be extensively tested. Experimental cell models, which are a suitable testing ground and the first line of empirical exploration of new biomaterials, must contain suitable cells that form the basis of biomaterial testing. To isolate a stable and suitable cell culture, many steps are required. The first and one of the most important steps is the collection of donor tissue, usually during a surgical procedure. Thus, the collection is the foundation for the success of cell isolation. This article explains the sources and neurosurgical procedures for obtaining brain tissue samples for cell isolation techniques, which are essential for biomaterial testing procedures.

CSIG-06. ELUCIDATING THE ROLE OF CO-OCCURRING MUTATIONS IN FGFR1-DRIVEN PEDIATRIC LOW-GRADE GLIOMA

Abstract Pediatric low-grade gliomas (pLGGs) have excellent survival, however, with current standard of care, most patients suffer lifelong severe sequalae. pLGGs are almost exclusively driven by single activating mutations in the mitogen-activated protein kinase (MAPK) pathway. As targeted molecular therapy clinical trials focusing on BRAF-altered pLGGs are showing promising results in early clinical trials, similar efforts are underway for FGFR1-altered tumors. In our cohort of FGFR1-4 altered gliomas, we observe frequent occurrence of a second alteration associated with activation of the MAPK or mammalian target of rapamycin (mTOR) pathways in pLGGs. Most commonly in the phosphatase NF1, PIK3CA, PIK3R1 PTPN11 or within the FGFR1 gene itself. However, the impact of second co-occurring mutations on therapeutic response and resistance has not been explored. To address this, we established mouse neural stem cell models over-expressing hot-spot mutated FGFR1 alone or in combination with a second alteration. Immunoblotting revealed that the addition of a second alteration attenuated phosphorylation of ERK, AKT and S6 and influenced cell proliferation both in normal growth conditions and in absence of growth factor. Treatment with an FGFR inhibitor (Infigratinib) showed reduced drug response in double mutant cells compared to hot-spot mutated FGFR1 alone. This was associated with less reduction of phosphorylation of ERK and S6 in the double mutant cells upon treatment. In conclusion, the presence of a second alteration influences proliferation and drug response in models of FGFR1-mutated pLGG, potentially by modulating MAPK and mTOR signaling.

Early Life Irradiation-Induced Hypoplasia and Impairment of Neurogenesis in the Dentate Gyrus and Adult Depression Are Mediated by MicroRNA- 34a-5p/T-Cell Intracytoplasmic Antigen-1 Pathway.

Early life radiation exposure causes abnormal brain development, leading to adult depression. However, few studies have been conducted to explore pre- or post-natal irradiation-induced depression-related neuropathological changes. Relevant molecular mechanisms are also poorly understood. We induced adult depression by irradiation of mice at postnatal day 3 (P3) to reveal hippocampal neuropathological changes and investigate their molecular mechanism, focusing on MicroRNA (miR) and its target mRNA and protein. P3 mice were irradiated by γ-rays with 5Gy, and euthanized at 1, 7 and 120 days after irradiation. A behavioral test was conducted before the animals were euthanized at 120 days after irradiation. The animal brains were used for different studies including immunohistochemistry, CAP-miRSeq, Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) and western blotting. The interaction of miR-34a-5p and its target T-cell intracytoplasmic antigen-1 (Tia1) was confirmed by luciferase reporter assay. Overexpression of Tia1 in a neural stem cell (NSC) model was used to further validate findings from the mouse model. Irradiation with 5 Gy at P3 induced depression in adult mice. Animal hippocampal pathological changes included hypoplasia of the infrapyramidal blade of the stratum granulosum, aberrant and impaired cell division, and neurogenesis in the dentate gyrus. At the molecular level, upregulation of miR-34a-5p and downregulation of Tia1 mRNA were observed in both animal and neural stem cell models. The luciferase reporter assay and gene transfection studies further confirmed a direct interaction between miR-34a-5p and Tia1. Our results indicate that the early life γ-radiation-activated miR-34a-5p/Tia1 pathway is involved in the pathogenesis of adult depression. This novel finding may provide a new therapeutic target by inhibiting the miR-34a-5p/Tia1 pathway to prevent radiation-induced pathogenesis of depression.

In vitro drug evaluation model of human iPS cell-derived neural cell by using novel gelatin fiber scaffold

In vitro drug evaluation model of human iPS cell-derived neural cell by using novel gelatin fiber scaffold

Proteomic profiling dataset of chemical perturbations in multiple biological backgrounds

While gene expression profiling has traditionally been the method of choice for large-scale perturbational profiling studies, proteomics has emerged as an effective tool in this context for directly monitoring cellular responses to perturbations. We previously reported a pilot library containing 3400 profiles of multiple perturbations across diverse cellular backgrounds in the reduced-representation phosphoproteome (P100) and chromatin space (Global Chromatin Profiling, GCP). Here, we expand our original dataset to include profiles from a new set of cardiotoxic compounds and from astrocytes, an additional neural cell model, totaling 5300 proteomic signatures. We describe filtering criteria and quality control metrics used to assess and validate the technical quality and reproducibility of our data. To demonstrate the power of the library, we present two case studies where data is queried using the concept of “connectivity” to obtain biological insight. All data presented in this study have been deposited to the ProteomeXchange Consortium with identifiers PXD017458 (P100) and PXD017459 (GCP) and can be queried at https://clue.io/proteomics.

Constrained plasticity reserve as a natural way to control frequency and weights in spiking neural networks

Biological neurons have adaptive nature and perform complex computations involving the filtering of redundant information. However, most common neural cell models, including biologically plausible, such as Hodgkin–Huxley or Izhikevich, do not possess predictive dynamics on a single-cell level. Moreover, the modern rules of synaptic plasticity or interconnections weights adaptation also do not provide grounding for the ability of neurons to adapt to the ever-changing input signal intensity. While natural neuron synaptic growth is precisely controlled and restricted by protein supply and recycling, weight correction rules such as widely used STDP are efficiently unlimited in change rate and scale. The present article introduces new mechanics of interconnection between neuron firing rate homeostasis and weight change through STDP growth bounded by abstract protein reserve, controlled by the intracellular optimization algorithm. We show how these cellular dynamics help neurons filter out the intense noise signals to help neurons keep a stable firing rate. We also examine that such filtering does not affect the ability of neurons to recognize the correlated inputs in unsupervised mode. Such an approach might be used in the machine learning domain to improve the robustness of AI systems.

The Beneficial Potential of Genetically Modified Stem Cells in the Treatment of Stroke: a Review.

The last two decades have witnessed a surge in investigations proposing stem cells as a promising strategy to treat stroke. Since growth factor release is considered as one of the most important aspects of cell-based therapy, stem cells over-expressing growth factors are hypothesized to yield higher levels of therapeutic efficiency. In pre-clinical studies of the last 15 years that were investigating the efficiency of stem cell therapy for stroke, a variety of stem cell types were genetically modified to over-express various factors. In this review we summarize the current knowledge on the therapeutic efficiency of stem cell-derived growth factors, encompassing techniques employed and time points to evaluate. In addition, we discuss several types of stem cells, including the recently developed model of epidermal neural crest stem cells, and genetically modified stem cells over-expressing specific factors, which could elevate the restorative potential of naive stem cells. The restorative potential is based on enhanced survival/differentiation potential of transplanted cells, apoptosis inhibition, infarct volume reduction, neovascularization or functional improvement. Since the majority of studies have focused on the short-term curative effects of genetically engineered stem cells, we emphasize the need to address their long-term impact.Graphical

Down-Regulation of Inpp5e Associated With Abnormal Ciliogenesis During Embryonic Neurodevelopment Under Inositol Deficiency.

The inositol polyphosphate-5-phosphatase E (Inpp5e) gene is located on chromosome 9q34.3. The enzyme it encodes mainly hydrolyzes the 5-phosphate groups of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5) P3) and phosphatidylinositol (4,5)-bisphosphate (PtdIns (4,5)P2), which are closely related to ciliogenesis and embryonic neurodevelopment, through mechanisms that are largely unknown. Here we studied the role of Inpp5e gene in ciliogenesis during embryonic neurodevelopment using inositol-deficiency neural tube defects (NTDs) mouse and cell models. Confocal microscopy and scanning electron microscope were used to examine the number and the length of primary cilia. The dynamic changes of Inpp5e expression in embryonic murine brain tissues were observed during Embryonic Day 10.5–13.5 (E 10.5–13.5). Immunohistochemistry, western blot, polymerase chain reaction (PCR) arrays were applied to detect the expression of Inpp5e and cilia-related genes of the embryonic brain tissues in inositol deficiency NTDs mouse. Real-time quantitative PCR (RT-qPCR) was used to validate the candidate genes in cell models. The levels of inositol and PtdIns(3,4) P2 were measured using gas chromatography-mass spectrometry (GC-MS) and enzyme linked immunosorbent assay (ELISA), respectively. Our results showed that the expression levels of Inpp5e gradually decreased in the forebrain tissues of the control embryos, but no stable trend was observed in the inositol deficiency NTDs embryos. Inpp5e expression in inositol deficiency NTDs embryos was significantly decreased compared with the control tissues. The expression levels of Inpp5e gene and the PtdIns (3,4) P2 levels were also significantly decreased in the inositol deficient cell model. A reduced number and length of primary cilia were observed in NIH3T3 cells when inositol deficient. Three important cilia-related genes (Ift80, Mkks, Smo) were down-regulated significantly in the inositol-deficient NTDs mouse and cell models, and Smo was highly involved in NTDs. In summary, these findings suggested that down-regulation of Inpp5e might be associated with abnormal ciliogenesis during embryonic neurodevelopment, under conditions of inositol deficiency.

Cold Atmospheric Plasma Elicits Neuroprotection Against Glutamate Excitotoxicity by Activating Cellular Antioxidant Defense

Neuroprotection is critical to rescue injured neural tissues and prevent secondary injuries but current clinical neuroprotection options are alarmingly limited. Following neural injury, reactive oxygen species (ROS) are aberrantly accumulated due to overactivation of glutamate, a major neurotransmitter in the vertebrate nervous system, leading to massive neural cell death. Antioxidant defense of neuronal cells is inherently repressed and poorly compensated by current pharmacological antioxidants. Here, we explore whether cold atmospheric plasma (CAP) with its ROS tuned near their physiological concentrations may be neuroprotective. Using a neural cell model, we show that a single treatment of a helium CAP jet for 4–10 s induces a pulsed elevation by 220% of reduced glutathione, the main ROS-scavenging system in cells, and substantially increase the viability of glutamate-damaged cells, demonstrating neuroprotection against glutamate excitotoxicity. By correlating peroxynitrite, hydrogen peroxide and nitric oxide produced by CAP to literature evidence of their neuroprotective activity when acting alone, we suggest that micromolar-level peroxynitrite and 30–70-µM hydrogen peroxide produced from the CAP jet trigger their known and dose-dependent pathways for activating the cellular antioxidant defense. Collectively, our data show that CAP offers a novel and potentially potent strategy to neuroprotection.

Three-Dimensional Human Neural Stem Cell Models to Mimic Heparan Sulfate Proteoglycans and the Neural Niche.

Heparan sulfate proteoglycans (HSPGs) are a diverse family of polysaccharides, consisting of a core protein with glycosaminoglycan (GAG) side chains attached. The heterogeneous GAG side-chain carbohydrates consist of repeating disaccharides, with each side chain possessing a specific sulfation pattern. It is the variable sulfation pattern that allows HSPGs to interact with numerous ligands including growth factors, cytokines, chemokines, morphogens, extracellular matrix (ECM) glycoproteins, collagens, enzymes, and lipases. HSPGs are classified according to their localization within an individual cell, and include the membrane bound syndecans (SDCs) and glypicans (GPCs), with perlecan, agrin, and type-XVIII collagen secreted into the ECM. The stem cell niche is defined as the environment that circumscribes stem cells when they are in their naïve state, and includes the ECM, which provides a complex contribution to various biological processes during development and throughout life. These contributions include facilitating cell adhesion, proliferation, migration, differentiation, specification, and cell survival. In contrast, HSPGs play an anticoagulant role in thrombosis through being present on the luminal surface of cells, while also playing roles in the stimulation and inhibition of angiogenesis, highlighting their varied and systemic roles in cellular control. To fully understand the complexities of cell-cell and cell-matrix interactions, three-dimensional (3D) models such as hydrogels offer researchers exciting opportunities, such as controllable 3D in vitro environments, that more readily mimic the in vivo/in situ microenvironment. This review examines our current knowledge of HSPGs in the stem cell niche, human stem cell models, and their role in the development of 3D models that mimic the in vivo neural ECM.

TORC1/2 kinase inhibition depletes glutathione and synergizes with carboplatin to suppress the growth of MYC-driven medulloblastoma.

Medulloblastoma is the most common malignant pediatric brain tumor. Tumors having high levels of c-MYC have the worst clinical prognosis, with only a minority of patients surviving. To address this unmet clinical need, we generated a human neural stem cell model of medulloblastoma that recapitulated the most aggressive subtype phenotypically and by mRNA expression profiling. An in silico analysis of these cells identified mTOR inhibitors as potential therapeutic agents. We hypothesized that the orally bioavailable TORC1/2 kinase inhibitor TAK228 would have activity against MYC-driven medulloblastoma. TAK228 inhibited mTORC1/2, decreased cell growth and caused apoptosis in high-MYC medulloblastoma cell lines. Comprehensive metabolic profiling of medulloblastoma orthotopic xenografts showed upregulation of glutathione compared to matched normal brain. TAK228 suppressed glutathione production. Because glutathione is required to detoxify platinum-containing chemotherapy, we hypothesized that TAK228 would cooperate with carboplatin in medulloblastoma. TAK228 synergized with carboplatin to inhibit cell growth and induce apoptosis and extended survival in orthotopic xenografts of high-MYC medulloblastoma. Brain-penetrant TORC1/2 inhibitors and carboplatin may be an effective combination therapy for high-risk medulloblastoma.

DNA repair pathways are altered in neural cell models of frataxin deficiency

Friedreich's ataxia (FRDA) is a hereditary and predominantly neurodegenerative disease caused by a deficiency of the protein frataxin (FXN). As part of the overall efforts to understand the molecular basis of neurodegeneration in FRDA, a new human neural cell line with doxycycline-induced FXN knockdown was established. This cell line, hereafter referred to as iFKD-SY, is derived from the human neuroblastoma SH-SY5Y and retains the ability to differentiate into mature neuron-like cells. In both proliferating and differentiated iFKD-SY cells, the induction of FXN deficiency is accompanied by increases in oxidative stress and DNA damage, reduced aconitase enzyme activity, higher levels of p53 and p21, activation of caspase-3, and subsequent apoptosis. More interestingly, FXN-deficient iFKD-SY cells exhibit an important transcriptional deregulation in many of the genes implicated in DNA repair pathways. The levels of some crucial proteins involved in DNA repair appear notably diminished. Furthermore, similar changes are found in two additional neural cell models of FXN deficit: primary cultures of FXN-deficient mouse neurons and human olfactory mucosa stem cells obtained from biopsies of FRDA patients. These results suggest that the deficiency of FXN leads to a down-regulation of DNA repair pathways that synergizes with oxidative stress to provoke DNA damage, which may be involved in the pathogenesis of FRDA. Thus, a failure in DNA repair may be considered a shared common molecular mechanism contributing to neurodegeneration in a number of hereditary ataxias including FRDA.

A Multiscale Approach to Axon and Nerve Stimulation Modeling: A Review.

Electrical nerve fiber stimulation is a technique widely used in prosthetics and rehabilitation, and its study from a computational point of view can be a useful instrument to support experimental tests. In the last years, there was an increasing interest in computational modeling of neural cells and numerical simulations on nerve fibers stimulation because of its usefulness in forecasting the effect of electrical current stimuli delivered to tissues through implanted electrodes, in the design of optimal stimulus waveforms based on the specific application (i.e., inducing limb movements, sensory feedback or physiological function restoring), and in the evaluation of the current stimuli properties according to the characteristics of the nerves surrounding tissue. Therefore, a review study on the main modeling and computational frameworks adopted to investigate peripheral nerve stimulation is an important instrument to support and drive future research works. To this aim, this paper deals with mathematical models of neural cells with a detailed description of ion channels and numerical simulations using finite element methods to describe the dynamics of electrical stimulation by implanted electrodes in peripheral nerve fibers. In particular, we evaluate different nerve cell models considering different ion channels present in neurons and provide a guideline on multiscale numerical simulations of electrical nerve fibers stimulation.