Involvement of Mitochondria in the Selective Response to Microsecond Pulsed Electric Fields on Healthy and Cancer Stem Cells in the Brain.

In the last few years, pulsed electric fields have emerged as promising clinical tools for tumor treatments. This study highlights the distinct impact of a specific pulsed electric field protocol, PEF-5 (0.3 MV/m, 40 µs, 5 pulses), on astrocytes (NHA) and medulloblastoma (D283) and glioblastoma (U87 NS) cancer stem-like cells (CSCs). We pursued this goal by performing ultrastructural analyses corroborated by molecular/omics approaches to understand the vulnerability or resistance mechanisms triggered by PEF-5 exposure in the different cell types. Electron microscopic analyses showed that, independently of exposed cells, the main targets of PEF-5 were the cell membrane and the cytoskeleton, causing membrane filopodium-like protrusion disappearance on the cell surface, here observed for the first time, accompanied by rapid cell swelling. PEF-5 induced different modifications in cell mitochondria. A complete mitochondrial dysfunction was demonstrated in D283, while a mild or negligible perturbation was observed in mitochondria of U87 NS cells and NHAs, respectively, not sufficient to impair their cell functions. Altogether, these results suggest the possibility of using PEF-based technology as a novel strategy to target selectively mitochondria of brain CSCs, preserving healthy cells.

Regionally specified human pluripotent stem cell-derived astrocytes exhibit different molecular signatures and functional properties.

Astrocytes display diverse morphologies in different regions of the central nervous system. Whether astrocyte diversity is attributable to developmental processes and bears functional consequences, especially in humans, is unknown. RNA-seq of human pluripotent stem cell-derived regional astrocytes revealed distinct transcript profiles, suggesting differential functional properties. This was confirmed by differential calcium signaling as well as effects on neurite growth and blood-brain barrier formation. Distinct transcriptional profiles and functional properties of human astrocytes generated from regionally specified neural progenitors under the same conditions strongly implicate the developmental impact on astrocyte diversity. These findings provide a rationale for renewed examination of regional astrocytes and their role in the pathogenesis of psychiatric and neurological disorders.

Alpha Tocopherol Loaded Polymeric Nanoparticles: Preparation, Characterizations, and In Vitro Assessments Against Oxidative Stress in Spinal Cord Injury Treatment.

Spinal cord injury (SCI) is characterized by mechanical injury or trauma to the spinal cord. Currently, SCI treatment requires extremely high doses of neuroprotective agents, which in turn, causes several adverse effects. To overcome these limitations, the present study focuses on delivery of a low but effective dose of a naturally occurring antioxidant, α-tocopherol (α-TP). Calcium alginate nanoparticles (CA-NP) and poly D,L-lactic-co-glycolic acid nanoparticles (PLGA-NP) prepared by ionotropic gelation and solvent evaporation technique had particle size of 21.9 ± 11.19 and 152.4 ± 10.6 nm, respectively. Surface morphology, surface charge, as well as particle size distribution of both nanoparticles were evaluated. Entrapment of α-TP into CA-NP and PLGA-NP quantified by UPLC showed entrapment efficiency of 4.00 ± 1.63% and 76.6 ± 11.4%, respectively. In vitro cytotoxicity profiles on human astrocyte-spinal cord (HA-sp) showed that blank CA-NP at high concentrations reduced the cell viability whereas blank PLGA-NP showed relatively safer cytotoxic profiles. In addition, PLGA nanoparticles encapsulated with α-TP (α-TP-PLGA-NP) in comparison to α-TP alone at high concentrations were less toxic. Pretreatment of HA-sp cells with α-TP-PLGA-NP showed two-fold higher anti-oxidative protection as compared to α-TP alone, when oxidative stress was induced by H2O2. In conclusion, CA-NP were found to be unsuitable for treatment of SCI due to their cytotoxicity. Comparatively, α-TP-PLGA-NP were safer and showed high degree of protection against oxidative stress than α-TP alone.

Physiological and Pathological Ageing of Astrocytes in the Human Brain.

Ageing is the greatest risk factor for dementia, although physiological ageing by itself does not lead to cognitive decline. In addition to ageing, APOE ε4 is genetically the strongest risk factor for Alzheimer's disease and is highly expressed in astrocytes. There are indications that human astrocytes change with age and upon expression of APOE4. As these glial cells maintain water and ion homeostasis in the brain and regulate neuronal transmission, it is likely that age- and APOE4-related changes in astrocytes have a major impact on brain functioning and play a role in age-related diseases. In this review, we will discuss the molecular and morphological changes of human astrocytes in ageing and the contribution of APOE4. We conclude this review with a discussion on technical issues, innovations, and future perspectives on how to gain more knowledge on astrocytes in the human ageing brain.

L-carnitine prevents ammonia-induced cytotoxicity and disturbances in intracellular amino acid levels in human astrocytes.

BACKGROUND AND AIM: L-carnitine (L-CA) has been used therapeutically to treat hepatic encephalopathy with hyperammonemia, but the mechanism by which L-CA contributes to ammonia detoxification in the brain is still unclear. Thus, the cytotoxicity and changes in intracellular amino acids (AAs) in astrocytes with hyperammonemia following L-CA administration were studied. METHODS: Human astrocytes were treated with ammonium chloride (NH4 Cl), L-CA or a mixture of NH4 Cl, and L-CA under defined conditions. Total intracellular reactive oxygen species and lactate dehydrogenase leakage were measured following different treatment periods. The intracellular levels of AAs in astrocytes were determined using metabolomic analysis. RESULTS: Intracellular total reactive oxygen species and lactate dehydrogenase leakage were significantly increased after treatment with NH4 Cl. In contrast, co-treatment with L-CA significantly inhibited the cytotoxic effects of NH4 Cl. The intracellular levels of almost all AAs involving glutamine and branched-chain AAs (BCAAs) were significantly increased in the NH4 Cl-treated cells compared with in the control cells; these changes in BCAA levels were reduced with L-CA co-treatment. Additionally, the level of 3-methyl-2-oxovaleric acid, which is a metabolite from isoleucine and plays a critical role in neurological damage, was significantly increased in the NH4 Cl-treated cells, but this metabolite was significantly decreased with L-CA co-treatment. CONCLUSION: L-CA protects human astrocytes from ammonia-induced acute cytotoxic effects and the increased intracellular levels of glutamine and BCAAs.

Do Evolutionary Changes in Astrocytes Contribute to the Computational Power of the Hominid Brain?

It is now well accepted that astrocytes are essential in all major nervous system functions of the rodent brain, including neurotransmission, energy metabolism, modulation of blood flow, ion and water homeostasis, and, indeed, higher cognitive functions, although the contribution of astrocytes in cognition is still in early stages of study. Here we review the most current research findings on human astrocytes, including their structure, molecular characterization, and functional properties. We also highlight novel tools that have been established for translational approaches to the comparative study of astrocytes from humans and experimental animals. Understanding the differences in astrocytes is essential to elucidate the contribution of astrocytes to normal physiology, cognitive processing and diverse pathologies of the central nervous system.

Calcium-sensing receptor antagonist (calcilytic) NPS 2143 specifically blocks the increased secretion of endogenous Aß42 prompted by exogenous fibrillary or soluble Aß25-35 in human cortical astrocytes and neurons-therapeutic relevance to Alzheimer's disease.

The "amyloid-ß (Aß) hypothesis" posits that accumulating Aß peptides (Aßs) produced by neurons cause Alzheimer's disease (AD). However, the Aßs contribution by the more numerous astrocytes remains undetermined. Previously we showed that fibrillar (f)Aß25-35, an Aß42 proxy, evokes a surplus endogenous Aß42 production/accumulation in cortical adult human astrocytes. Here, by using immunocytochemistry, immunoblotting, enzymatic assays, and highly sensitive sandwich ELISA kits, we investigated the effects of fAß25-35 and soluble (s)Aß25-35 on Aß42 and Aß40 accumulation/secretion by human cortical astrocytes and HCN-1A neurons and, since the calcium-sensing receptor (CaSR) binds Aßs, their modulation by NPS 2143, a CaSR allosteric antagonist (calcilytic). The fAß25-35-exposed astrocytes and surviving neurons produced, accumulated, and secreted increased amounts of Aß42, while Aß40 also accrued but its secretion was unchanged. Accordingly, secreted Aß42/Aß40 ratio values rose for astrocytes and neurons. While slightly enhancing Aß40 secretion by fAß25-35-treated astrocytes, NPS 2143 specifically suppressed the fAß25-35-elicited surges of endogenous Aß42 secretion by astrocytes and neurons. Therefore, NPS 2143 addition always kept Aß42/Aß40 values to baseline or lower levels. Mechanistically, NPS 2143 decreased total CaSR protein complement, transiently raised proteasomal chymotrypsin activity, and blocked excess NO production without affecting the ongoing increases in BACE1/ß-secretase and γ-secretase activity in fAß25-35-treated astrocytes. Compared to fAß25-35, sAß25-35 also stimulated Aß42 secretion by astrocytes and neurons and NPS 2143 specifically and wholly suppressed this effect. Therefore, since NPS 2143 thwarts any Aß/CaSR-induced surplus secretion of endogenous Aß42 and hence further vicious cycles of Aß self-induction/secretion/spreading, calcilytics might effectively prevent/stop the progression to full-blown AD.

Phase-amplitude coupling detection and analysis of human 2-dimensional neural cultures in multi-well microelectrode array in vitro.

BACKGROUND: Human induced pluripotent stem cell (hiPSC)- derived neurons offer the possibility of studying human-specific neuronal behaviors in physiologic and pathologic states in vitro. It is unclear whether cultured neurons can achieve the fundamental network behaviors required to process information in the brain. Investigating neuronal oscillations and their interactions, as occurs in cross-frequency coupling (CFC), addresses this question. NEW METHODS: We examined whether networks of two-dimensional (2D) cultured hiPSC-derived cortical neurons grown with hiPSC-derived astrocytes on microelectrode array plates recapitulate the CFC that is present in vivo. We employed the modulation index method for detecting phase-amplitude coupling (PAC) and used offline spike sorting to analyze the contribution of single neuron spiking to network behavior. RESULTS: We found that PAC is present, the degree of PAC is specific to network structure, and it is modulated by external stimulation with bicuculline administration. Modulation of PAC is not driven by single neurons, but by network-level interactions. COMPARISON WITH EXISTING METHODS: PAC has been demonstrated in multiple regions of the human cortex as well as in organoids. This is the first report of analysis demonstrating the presence of coupling in 2D cultures. CONCLUSION: CFC in the form of PAC analysis explores communication and integration between groups of neurons and dynamical changes across networks. In vitro PAC analysis has the potential to elucidate the underlying mechanisms as well as capture the effects of chemical, electrical, or ultrasound stimulation; providing insight into modulation of neural networks to treat nervous system disorders in vivo.

Cortical Astrocyte Progenitors and Astrocytes from Human Pluripotent Stem Cells.

Astrocytes coordinate several homeostatic processes of the central nervous system and play essential roles for normal brain development and response to disease conditions. Protocols for the conversion of human induced pluripotent stem cells (hiPSCs) into mature astrocytes have opened to the generation of in vitro systems to explore astrocytes' functions in living human cell contexts and patient-specific settings. In this study, we present an optimized monolayer procedure to commit hiPSC-derived cortical progenitors into enriched populations of cortical astrocyte progenitor cells (CX APCs) that can be further amplified and efficiently differentiated into mature astrocytes. Our optimized system provides a valid tool to explore the role of these cells in neurodevelopmental and neuropsychiatric diseases, opening it up to applications in drug development and biomarkers discovery/validation.

Integrating Primary Astrocytes in a Microfluidic Model of the Blood-Brain Barrier.

An in vitro blood-brain barrier (BBB) model must be highly reproducible and imitate as much as possible the properties of the in vivo environment, from both the functional and anatomical point of view. In our latest work, a BBB prototype was implemented through the use of human primary brain cells and then integrated in a microfluidic platform (Lauranzano et al., Adv Biosyst 3:e1800335, 2019). Here we describe, step by step, the setting of a customized bio-mimetic platform, which uses human brain endothelial cells and primary astrocytic cells to allow the study of the complex interactions between the immune system and the brain in healthy and neuroinflammatory conditions. The model can be exploited to investigate the neuroimmune communication at the blood-brain interface and to examine the transmigration of patient-derived lymphocytes in order to envisage cutting-edge strategies to restore barrier integrity and block the immune cell influx into the CNS.

Overexpression of α-Synuclein Reorganises Growth Factor Profile of Human Astrocytes.

Misfolding and accumulation of aberrant α-synuclein in the brain is associated with the distinct class of neurodegenerative diseases known as α-synucleinopathies, which include Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Pathological changes in astrocytes contribute to all neurological disorders, and astrocytes are reported to possess α-synuclein inclusions in the context of α-synucleinopathies. Astrocytes are known to express and secrete numerous growth factors, which are fundamental for neuroprotection, synaptic connectivity and brain metabolism; changes in growth factor secretion may contribute to pathobiology of neurological disorders. Here we analysed the effect of α-synuclein overexpression in cultured human astrocytes on growth factor expression and release. For this purpose, the intracellular and secreted levels of 33 growth factors (GFs) and 8 growth factor receptors (GFRs) were analysed in cultured human astrocytes by chemiluminescence-based western/dot blot. Overexpression of human α-synuclein in cultured foetal human astrocytes significantly changes the profile of GF production and secretion. We found that human astrocytes express and secrete FGF2, FGF6, EGF, IGF1, AREG, IGFBP2, IGFBP4, VEGFD, PDGFs, KITLG, PGF, TGFB3 and NTF4. Overexpression of human α-synuclein significantly modified the profile of GF production and secretion, with particularly strong changes in EGF, PDGF, VEGF and their receptors as well as in IGF-related proteins. Bioinformatics analysis revealed possible interactions between α-synuclein and EGFR and GDNF, as well as with three GF receptors, EGFR, CSF1R and PDGFRB.

Efficient and rapid conversion of human astrocytes and ALS mouse model spinal cord astrocytes into motor neuron-like cells by defined small molecules.

BACKGROUND: Motor neuron degeneration or loss in the spinal cord is the characteristic phenotype of motor neuron diseases or spinal cord injuries. Being proliferative and located near neurons, astrocytes are considered ideal cell sources for regenerating neurons. METHODS: We selected and tested different combinations of the small molecules for inducing the conversion of human and mouse astrocytes into neurons. Microscopic imaging and immunocytochemistry analyses were used to characterize the morphology and phenotype of the induced neurons while RT-qPCR was utilized to analyze changes in gene expression. In addition, whole-cell patch-clamp recordings were measured to examine the electrophysiological properties of induced neurons. RESULTS: The results showed that human astrocytes could be rapidly and efficiently converted into motor neuron-like cells by treatment with defined small molecules, with a yield of over 85% motor neuron-like cells attained. The induced motor neuron-like cells expressed the pan-neuronal markers TUJ1, MAP2, NeuN, and Synapsin 1 and motor neuron markers HB9, ISL1, CHAT, and VAChT. During the conversion process, the cells did not pass through a proliferative neural progenitor cell intermediate. The induced motor neurons were functional, showing the electrophysiological properties of neurons. The same chemical cocktail could induce spinal cord astrocytes from an amyotrophic lateral sclerosis mouse model carrying a SOD1 mutation to become motor neuron-like cells that exhibited a decrease in cell survival and an increase in oxidative stress compared to that observed in wild-type MNs derived from healthy mice. Moreover, the chemical induction reduced oxidative stress in the mutant astrocytes. CONCLUSION: The results of the present study demonstrated the feasibility of chemically converting human and mouse astrocytes into motor neuron-like cells that are useful for neurodegenerative disease modeling and regenerative medicine.

ß2-Adrenergic Receptor Stimulation Upregulates Cx43 Expression on Glioblastoma Multiforme and Olfactory Ensheathing Cells.

Glioblastoma multiforme (GBM) is described as an invasive astrocytic tumor in adults. Despite current standard treatment approaches, the outcome of GBM remains unfavorable. The downregulation of connexin 43 (Cx43) expression is one of the molecular transformations in GBM cells. The Cx43 levels and subsequently gap junctional intercellular communication (GJIC) have an important role in the efficient transfer of cytotoxic drugs to whole tumor cells. As shown in our previous study, the stimulation of the ß2-adrenergic receptor (ß2-AR) leads to the modulation of Cx43 expression level in the GBM cell line. Here we further examine the effect of clenbuterol hydrochloride as a selective ß2-AR agonist on the Cx43 expression in human GBM-derived astrocyte cells and human olfactory ensheathing cells (OECs) as a potent vector for future gene therapy. In this experiment, first we established a primary culture of astrocytes from GBM samples and verified the purity using immunocytofluorescent staining. Western blot analysis was performed to evaluate the Cx43 protein level. Our western blot findings reveal that clenbuterol hydrochloride upregulates the Cx43 protein level in both primary human astrocyte cells and human OECs. Conversely, ICI 118551 as a ß2-AR antagonist inhibits these effects. Moreover, clenbuterol hydrochloride increases the Cx43 expression in primary human astrocyte cells and OECs co-culture systems, and ICI 118551 reverses these effects. To confirm the western blot results, immunocytofluorescent staining was performed to evaluate the ß2-AR agonist effect on Cx43 expression. Our immunocytofluorescent results supported western blot analysis in primary human astrocyte cells and the OECs co-culture system. The results of this study suggest that the activation of ß2-AR with regard to Cx43 protein levels enhancement in GBM cells and OECs might be a promising approach for GBM treatment in the future.

Involvement of monocarboxylate transporter 1 (SLC16A1) in the uptake of l-lactate in human astrocytes.

PURPOSE: Astrocytes, the most abundant glial cells in the central nervous system (CNS), help neurons survive. Monocarboxylate transporters (MCTs) are reported to transport l-lactate, which is important for CNS physiology and cognitive function. However, it remains unclear which MCT isoform is functionally expressed by human astrocytes. The aim of this study was to establish the contribution of each MCT isoform to l-lactate transport in human astrocytes. METHODS: The function of l-lactate transport was studied using NHA cells as a human astrocyte model and radiolabeled l-lactate. The expression of MCT in human astrocytes was detected by immunohistochemistry staining. RESULTS: The cellular uptake of l-lactate was found to be pH- and concentration-dependent with a Km value for l-lactate uptake of 0.64mM. This Km was similar to what has been previously established for MCT1-mediated l-lactate uptake. α-Cyano-4- hydroxycinnamate (CHC) and 5-oxoproline, which are both MCT1 inhibitors, were found to significantly inhibit the uptake of l-lactate, suggesting MCT1 is primarily responsible for l-lactate transport. Moreover, MCT1 protein was expressed in human astrocytes. CONCLUSION: pH-dependent l-lactate transport is mediated by MCT1 in human astrocytes.

The investigation of the pyrethroid insecticide lambda-cyhalothrin (LCT)-affected Ca2+ homeostasis and -activated Ca2+-associated mitochondrial apoptotic pathway in normal human astrocytes: The evaluation of protective effects of BAPTA-AM (a selective Ca2+ chelator).

Exposure to insecticides has been found to have deleterious effects on human health. Lambda-cyhalothrin (LCT), a mixture of isomers of cyhalothrin, is a pyrethroid insecticide routinely used in pest control programs. LCT was reported to cause neurotoxic effects in various models. However, the mechanism of underlying effect of LCT on cytotoxicity in normal human brain cells is still elusive. This study examined whether LCT affected Ca2+ homeostasis and Ca2+-related physiology in Gibco® Human Astrocytes (GHA cells), and explored whether BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid), a selective Ca2+ chelator, has protective effects on LCT-treated GHA cells. The data show that LCT (10-15 µM) concentration-dependently induced cytotoxicity in both GHA cells and DI TNC1 normal rat astrocytes but only induced intracellular Ca2+ concentration ([Ca2+]i) rises in GHA cells. In terms of Ca2+ signaling in GHA cells, LCT-induced [Ca2+]i rises were reduced by removing extracellular Ca2+ and were inhibited by store-operated Ca2+ channel modulators (2-APB, econazole or SKF96365). In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished LCT-induced [Ca2+]i rises. Conversely, incubation with LCT abolished thapsigargin-induced [Ca2+]i rises. Regarding cytotoxicity, LCT evoked apoptosis by regulating apoptotic protein expressions (Bax, BCl-2, cleaved caspase-9/-3). This apoptotic response was significantly inhibited by prechelating cytosolic Ca2+ with BAPTA-AM. Together, in GHA cells, LCT induced [Ca2+]i rises by inducing Ca2+ entry via store-operated Ca2+ channels and Ca2+ release from the endoplasmic reticulum. Moreover, BAPTA-AM has a protective effect on inhibiting LCT-activated mitochondrial apoptotic pathway. This study provided new insights into the molecular protective mechanism of LCT-induced cytotoxicity in normal human astrocytes.

3D Culture Method for Alzheimer's Disease Modeling Reveals Interleukin-4 Rescues Aß42-Induced Loss of Human Neural Stem Cell Plasticity.

Neural stem cells (NSCs) constitute an endogenous reservoir for neurons that could potentially be harnessed for regenerative therapies in disease contexts such as neurodegeneration. However, in Alzheimer's disease (AD), NSCs lose plasticity and thus possible regenerative capacity. We investigate how NSCs lose their plasticity in AD by using starPEG-heparin-based hydrogels to establish a reductionist 3D cell-instructive neuro-microenvironment that promotes the proliferative and neurogenic ability of primary and induced human NSCs. We find that administration of AD-associated Amyloid-ß42 causes classical neuropathology and hampers NSC plasticity by inducing kynurenic acid (KYNA) production. Interleukin-4 restores NSC proliferative and neurogenic ability by suppressing the KYNA-producing enzyme Kynurenine aminotransferase (KAT2), which is upregulated in APP/PS1dE9 mouse model of AD and in postmortem human AD brains. Thus, our culture system enables a reductionist investigation of regulation of human NSC plasticity for the identification of potential therapeutic targets for intervention in AD.

MiR-140/BDNF axis regulates normal human astrocyte proliferation and LPS-induced IL-6 and TNF-α secretion.

Reactive astrocyte proliferation after spinal cord injury (SCI) contributes to glial scar formation that impedes axonal regeneration. The mechanisms underlying astrocyte proliferation upon injury remain partially understood. MicroRNAs (miRNAs) function as a major class of post-transcriptional gene expression regulators that participate in many biological processes. In this study, we focused on the functional role of miR-140 in normal human astrocyte (NHA) cell proliferation. Ectopic miR-140 expression significantly inhibited NHA cell viability and proliferation; miR-140 inhibition exerted the opposite function. Commonly, miRNAs exert functions through targeting downstream genes to inhibit their expression. In the present study, brain-derived neurotrophic factor (BDNF), a regulator of astrocyte proliferation and differentiation, confirmed as a direct target of miR-140 in NHA. Through binding to the 3'UTR of BDNF, miR-140 inhibited BDNF expression. BDNF overexpression significantly promoted NHA cell viability and proliferation; the regulatory effect of miR-140/BDNF on NHA proliferation was mediated by PI3K/AKT pathway. Moreover, we evaluated the functional role of miR-140 in Lipopolysaccharide (LPS)-induced in vitro injury model of astroglial cultures; a significantly up-regulated BDNF, interleukin (IL)-6 and tumor necrosis factor (TNF)-α expression in response to LPS stimulation was observed. After ectopic miR-140 expression, the promotive effect of LPS on BDNF, IL-6 and TGF-α expression was partially restored. Taken together, miR-140/BDNF axis regulates NHA proliferation through PI3K/AKT pathway; miR-140 could inhibit BDNF, IL-6 and TGF-α expression in LPS-induced in vitro injury model. MiR-140/BDNF might serve as a promising target in strategy against reactive astrocyte proliferation after SCI.

MicroRNA-211/BDNF axis regulates LPS-induced proliferation of normal human astrocyte through PI3K/AKT pathway.

Spinal cord injury (SCI) makes a major contribution to disability and deaths worldwide. Reactive astrogliosis, a typical feature after SCI, which undergoes varying molecular and morphological changes, is ubiquitous but poorly understood. Reactive astrogliosis contributes to glial scar formation that impedes axonal regeneration. Brain-derived neurotrophic factor (BDNF), a well-established neurotrophic factor, exerts neuroprotective and growth-promoting effects on a variety of neuronal populations after injury. In the present study, by using LPS-induced in vitro injury model of astroglial cultures, we observed a high expression of interleukin (IL)-6, IL-1ß, and BDNF in LPS-stimulated normal human astrocytes (NHAs). BDNF significantly promoted NHA proliferation. Further, online tools were employed to screen the candidate miRNAs which might directly target BDNF to inhibit its expression. Amongst the candidate miRNAs, miR-211 expression was down-regulated by LPS stimulation in a dose-dependent manner. Through direct targetting, miR-211 inhibited BDNF expression. Ectopic miR-211 expression significantly suppressed NHA proliferation, as well as LPS-induced activation of PI3K/Akt pathway. In contrast, inhibition of miR-211 expression significantly promoted NHA proliferation and LPS-induced activation of PI3K/Akt pathway. Taken together, miR-211/BDNF axis regulates LPS-induced NHA proliferation through PI3K/AKT pathway; miR-211/BDNF might serve as a promising target in the strategy against reactive astrocyte proliferation after SCI.

Complement-dependent and -independent aquaporin 4-antibody-mediated cytotoxicity in human astrocytes: Pathogenetic implications in neuromyelitis optica.

BACKGROUND: Neuromyelitis optica (NMO) is an inflammatory disease caused by the aquaporin (AQP)-4-antibody. Pathological studies on NMO have revealed extensive astrocytic damage, as evidenced by the loss of AQP4 and glial fibrillary acidic protein (GFAP), specifically in perivascular regions with immunoglobulin and complement depositions, although other pathological patterns, such as a loss of AQP4 without astrocyte destruction and clasmatodendrosis, have also been observed. Previous studies have shown that complement-dependent antibody-mediated astrocyte lysis is likely a major pathomechanism in NMO. However, there are also data to suggest antibody-mediated astrocyte dysfunction in the absence of complement. Thus, the importance of complement inhibitory proteins in complement-dependent AQP4-antibody-mediated astrocyte lysis in NMO is unclear. In most of the previous studies, the complement and target cells (astrocytes or AQP4-transfected cells) were derived from different species; however, the complement inhibitory proteins that are expressed on the cell surface cannot protect themselves against complement-dependent cytolysis unless the complements and complement inhibitory proteins are from the same species. To resolve these issues, we studied human astrocytes in primary culture treated with AQP4-antibody in the presence or absence of human complement and examined the effect of complement inhibitory proteins using small interfering RNA (siRNA). METHODS: Purified IgG (10 mg/mL) was obtained from 5 patients with AQP4-antibody-positive NMO, 3 patients with multiple sclerosis (MS), and 3 healthy controls. Confluent human astrocytes transfected with Venus-M1-AQP4-cDNA were incubated with IgG (5% volume). After washing, we cultured the cells with human complements with or without heat inactivation. We observed time-lapse morphological and immunohistochemical changes using a fluorescence microscope. We also evaluated cytotoxicity using a propidium iodide (PI) kit and the lactate dehydrogenase (LDH) assay. RESULT: AQP4-antibody alone caused clustering and degradation followed by endocytosis of membraneous AQP4, thereby resulting in decreased cellular adherence and the shrinkage of astrocytic processes. However, these changes were partially reversed by the removal of IgG in culture. In contrast, following the application of AQP4-antibody and non-heated human complements, the cell bodies and nuclei started to swell. At 3 h, most of the astrocytes had lost mobility and adherence and were eventually destroyed or had swollen and were then destroyed. In addition, the remaining adherent cells were mostly PI-positive, indicating necrosis. Astrocyte lysis caused by rabbit complement occurred much faster than did cell lysis with human complement. However, the cell lysis was significantly enhanced by the transfection of astrocytes with siRNA against human CD55 and CD59, which are major complement inhibitory proteins on the astrocyte membrane. AQP4-antibody-negative IgG in MS or control did not induce such changes. CONCLUSION: Taken together, these findings suggest that both complement-dependent and complement-independent AQP4-antibody-mediated astrocytopathies may operate in NMO, potentially contributing to diverse pathological patterns. Our results also suggest that the effect of complement inhibitory proteins should be considered when evaluating AQP4-antibody-mediated cytotoxicity in AQP4-expressing cells.

Human astrocytic grid networks patterned in parylene-C inlayed SiO2 trenches.

Recent literature suggests that glia, and in particular astrocytes, should be studied as organised networks which communicate through gap junctions. Astrocytes, however, adhere to most surfaces and are highly mobile cells. In order to study, such organised networks effectively in vitro it is necessary to influence them to pattern to certain substrates whilst being repelled from others and to immobilise the astrocytes sufficiently such that they do not continue to migrate further whilst under study. In this article, we demonstrate for the first time how it is possible to facilitate the study of organised patterned human astrocytic networks using hNT astrocytes in a SiO2 trench grid network that is inlayed with the biocompatible material, parylene-C. We demonstrate how the immobilisation of astrocytes lies in the depth of the SiO2 trench, determining an optimum trench depth and that the optimum patterning of astrocytes is a consequence of the parylene-C inlay and the grid node spacing. We demonstrate high fidelity of the astrocytic networks and demonstrate that functionality of the hNT astrocytes through ATP evoked calcium signalling is also dependent on the grid node spacing. Finally, we demonstrate that the location of the nuclei on the grid nodes is also a function of the grid node spacing. The significance of this work, is to describe a suitable platform to facilitate the study of hNT astrocytes from the single cell level to the network level to improve knowledge and understanding of how communication links to spatial organisation at these higher order scales and trigger in vitro research further in this area with clinical applications in the area of epilepsy, stroke and focal cerebral ischemia.