A primary neural cell culture model to study neuron, astrocyte, and microglia interactions in neuroinflammation.

BackgroundInteractions between neurons, astrocytes, and microglia critically influence neuroinflammatory responses to insult in the central nervous system. In vitro astrocyte and microglia cultures are powerful tools to study specific molecular pathways involved in neuroinflammation; however, in order to better understand the influence of cellular crosstalk on neuroinflammation, new multicellular culture models are required.MethodsPrimary cortical cells taken from neonatal rats were cultured in a serum-free "tri-culture" medium formulated to support neurons, astrocytes, and microglia, or a "co-culture" medium formulated to support only neurons and astrocytes. Caspase 3/7 activity and morphological changes were used to quantify the response of the two culture types to different neuroinflammatory stimuli mimicking sterile bacterial infection (lipopolysaccharide (LPS) exposure), mechanical injury (scratch), and seizure activity (glutamate-induced excitotoxicity). The secreted cytokine profile of control and LPS-exposed co- and tri-cultures were also compared.ResultsThe tri-culture maintained a physiologically relevant representation of neurons, astrocytes, and microglia for 14 days in vitro, while the co-cultures maintained a similar population of neurons and astrocytes, but lacked microglia. The continuous presence of microglia did not negatively impact the overall health of the neurons in the tri-culture, which showed reduced caspase 3/7 activity and similar neurite outgrowth as the co-cultures, along with an increase in the microglia-secreted neurotrophic factor IGF-1 and a significantly reduced concentration of CX3CL1 in the conditioned media. LPS-exposed tri-cultures showed significant astrocyte hypertrophy, increase in caspase 3/7 activity, and the secretion of a number of pro-inflammatory cytokines (e.g., TNF, IL-1α, IL-1β, and IL-6), none of which were observed in LPS-exposed co-cultures. Following mechanical trauma, the tri-culture showed increased caspase 3/7 activity, as compared to the co-culture, along with increased astrocyte migration towards the source of injury. Finally, the microglia in the tri-culture played a significant neuroprotective role during glutamate-induced excitotoxicity, with significantly reduced neuron loss and astrocyte hypertrophy in the tri-culture.ConclusionsThe tri-culture consisting of neurons, astrocytes, and microglia more faithfully mimics in vivo neuroinflammatory responses than standard mono- and co-cultures. This tri-culture can be a useful tool to study neuroinflammation in vitro with improved accuracy in predicting in vivo neuroinflammatory phenomena

Sustained synchronized neuronal network activity in a human astrocyte co-culture system

Impaired neuronal network function is a hallmark of neurodevelopmental and neurodegenerative disorders such as autism, schizophrenia, and Alzheimer's disease and is typically studied using genetically modified cellular and animal models. Weak predictive capacity and poor translational value of these models urge for better human derived in vitro models. The implementation of human induced pluripotent stem cells (hiPSCs) allows studying pathologies in differentiated disease-relevant and patient-derived neuronal cells. However, the differentiation process and growth conditions of hiPSC-derived neurons are non-trivial. In order to study neuronal network formation and (mal) function in a fully humanized system, we have established an in vitro co-culture model of hiPSC-derived cortical neurons and human primary astrocytes that recapitulates neuronal network synchronization and connectivity within three to four weeks after final plating. Live cell calcium imaging, electrophysiology and high content image analyses revealed an increased maturation of network functionality and synchronicity over time for co-cultures compared to neuronal monocultures. The cells express GABAergic and glutamatergic markers and respond to inhibitors of both neurotransmitter pathways in a functional assay. The combination of this co-culture model with quantitative imaging of network morphofunction is amenable to high throughput screening for lead discovery and drug optimization for neurological diseases

Exploiting 3D differentiation of human stem cells

"Neurological disorders are a major public health problem and are expected to rise dramatically together with the higher life expectancy and the shift towards an ageing society. Current therapeutic options can only ameliorate some of the symptoms and there are no effective treatments to target pathological mechanisms and stop disease progression. The human brain complexity hampers the understanding of the brain functioning at the molecular, cellular, and pathophysiological levels for many neurological disorders. This highlights the need for new brain models, which can contribute to unveil molecular mechanisms of neurological disorders, identify therapeutic targets and evaluate preclinically new therapies in a more adequate and predictive basis, withstanding its successful translation to the clinics. Despite their undeniable value, traditional animal models diverge from humans at biochemical and genetic levels. Moreover, 2D in vitro cell-based models do not mimic important aspects of brain cellular heterogeneity, architecture and microenvironment (...)".N/

The comparative effects between tocotrieonol-rich fraction (TRF) and α-tocopherol on glutamate toxicity in neuron-astrocyte mono- and co-culture systems

Background: Vitamin E, which can be categorized into tocotrienols and tocopherols, is known to protect cells from glutamate neurotoxicity. Studies have shown that tocotrienol-rich fraction (TRF) protecting the brain against oxidative damage more efficient than α-tocopherol. The role of astrocyte in promoting neuronal survival and recovery after glutamate neurotoxicity is also increasingly appreciated. Aims: To elucidate the effects of TRF and α-tocopherol and the synergism between astrocyte and neuron against glutamate neurotoxicity. Methods: Astrocyte and neuron were subjected to glutamate injury followed by TRF and α-tocopherol treatments (100 – 300 ng/ml). Effects of TRF and α-tocopherol on nerve cell viability and glutathione contents against glutamate toxicity were examined. The synergism between astrocyte and neuron was elucidated through co-culture model. Statistical analysis was performed using one way ANOVA. Results: Both TRF and α-tocopherol improved approximately 10% of glutamate-injured astrocyte and neuronal cell viability. In co-culture model, TRF and α-tocopherol provided nearly complete protection from glutamate toxicity. Besides, TRF and α-tocopherol treatments significantly restored at least 20% of glutathione contents in glutamate-injured neurons. In the presence of astrocyte, 300 ng/ml TRF and α-tocopherol completely restored glutathione contents in glutamate-injured neuron. Conclusions: TRF and α-tocopherol had shown promising neuroprotective effects in astrocyte and neuron from glutamate toxicity. Great scavenging effect of both TRF and α-tocopherol against glutamate toxicity was observed in neuron. Similar protective effects between TRF and α-tocopherol were observed. Co-culture model demonstrated the synergistic properties between neuron and astrocyte. Supplementation of TRF and α-tocopherol in co-culture further improved the recovery process

뉴런, 성상세포 및 HEK293T 세포에서의 시험관 내 eGRASP 발현에 대한 연구

학위논문 (석사) -- 서울대학교 대학원 : 자연과학대학 협동과정 뇌과학전공, 2021. 2. 강봉균.Studies on astrocytes have been focused on unveiling its interplay with neurons to understand the physiological properties of nerve systems, leading to both fundamental interests and clinical applications including the origins of neurodegenerative diseases. In terms of synaptic transmission, major role of astrocytes is forming ‘tripartite synapse’ among postsynaptic neurons and presynaptic neurons. However, progress in the understanding of interactions among tripartite synapses has been limited to topical observation for lack of effective technologies to visualize synapses. Recently, the dual-eGRASP was developed to label synapses of targeted neurons in the mammalian brain. The technique made it possible to compare the activity- or region-dependent strength of synapses, potentially providing a tool to elucidate the role of neuronal connectivity in learning and memory. Here, I verified the eGRASP signals to be present in in vitro rodent primary neurons and astrocytes, expanding the applications of previously published dual-eGRASP methodology. The eGRASP signals were expressed among various cells including neurons, astrocytes, and HEK293T cells, regardless of transfection methods. Especially, the signals appeared in the form of faces at contacts between cells: dendritic shafts and adjoining astrocytes or HEK293T cells. The results substantiate that the in vitro eGRASP provides a promising method to elucidate not only physiologies of nerve systems in controlled environment, but also of any cellular systems with an arbitrary level of external control.성상세포와 뉴런 사이의 상호 작용 연구는 신경계의 생리적 특성을 이해하고 신경 퇴행성 질환을 비롯한 임상적 측면에 적용하기 위해 진행되어 왔다. 시냅스 신호 전달의 측면에서 성상세포의 주요 기능은 시냅스 후 뉴런과 시냅스 전 뉴런 사이에서 '삼자 간 시냅스'를 형성하는 것이다. 그러나 시냅스를 효율적으로 시각화하는 기술이 부족하였기에 삼자 간 시냅스에 대한 이해는 국소적인 관찰에 그쳤다. 최근 dual-eGRASP 기술이 개발되어 포유류 뇌에 있는 표적 뉴런의 시냅스 표시가 가능해졌다. 이 기법은 시냅스의 활성 상태나 지역에 따른 강도의 비교를 가능하게 했으며, 학습과 기억에서 뉴런 간 연결이 어떤 기능을 하는지 설명할 도구를 제공했다. 본 학위논문은 dual eGRASP 방법론의 적용을 확대하여 시험관 내에서 일차 뉴런과 성상세포 eGRASP 신호를 검증했다. eGRASP는 형질 전환 방법에 상관없이 뉴런, 성상세포, HEK293T 세포 등 다양한 세포 사이에서 발현됐다. 신호는 수상돌기의 축과 인접한 성상세포ᆞHEK293T 세포 사이의 접촉 사이에서 면의 형태로 나타났다. 이러한 결과는 시험관 내 eGRASP가 통제된 환경의 신경계의 생리학 연구와 더불어 다양한 세포의 생리학을 설명할 유망한 방법을 제공한다는 것을 입증한다.List of Figures ５ Abstract ６ Introduction ８ Materials and methods １３ Results ２１ Figures ３０ Discussion ５１ References ５６ Abstract in Korean ６２Maste

Biomimetic Modeling of Preeclamptic Conditions

Background: •Preeclampsia is a condition often diagnosed around 20 weeks of pregnancy •Preeclampsia results in 18% of US maternal deaths and 15% of premature US births •Results in high blood pressure, headaches, nausea, and breathing difficulties Hypothesis: Placental stiffness shares a direct correlation with the adverse progression of preeclampsia. Aims/Objectives: •Characterize the relationship between stiffness and alterations to placental cell morphology, metabolism, and function Morphology: Once the plates have been seeded they are monitored for several days, when they reach adequate confluence they are imaged for changes in morphology. The images show cell elongation with increasing stiffness

Rapid astrocyte-dependent facilitation amplifies multi-vesicular release in hippocampal synapse

Synaptic facilitation is a major form of short-term plasticity typically driven by an increase in residual presynaptic calcium. Using near-total internal reflection fluorescence (near-TIRF) imaging of single vesicle release in cultured hippocampal synapses, we demonstrate a distinctive, release-dependent form of facilitation in which probability of vesicle release is higher following a successful glutamate release event than following a failure. This phenomenon has an onset of ≤500 ms and lasts several seconds, resulting in clusters of successful release events. The release-dependent facilitation requires neuronal contact with astrocytes and astrocytic glutamate uptake by EAAT1. It is not observed in neurons grown alone or in the presence of astrocyte-conditioned media. This form of facilitation dynamically amplifies multi-vesicular release. Facilitation-evoked release events exhibit spatial clustering and have a preferential localization toward the active zone center. These results uncover a rapid astrocyte-dependent form of facilitation acting via modulation of multi-vesicular release and displaying distinctive spatiotemporal properties

Picomolar concentrations of oligomeric alpha-synuclein sensitizes TLR4 to play an initiating role in Parkinson’s disease pathogenesis

Funder: Alzheimer’s Research UK; doi: http://dx.doi.org/10.13039/501100002283Abstract: Despite the wealth of genomic and transcriptomic data in Parkinson’s disease (PD), the initial molecular events are unknown. Using LD score regression analysis, we show significant enrichment in PD heritability within regulatory sites for LPS-activated monocytes and that TLR4 expression is highest within human substantia nigra, the most affected brain region, suggesting a role for TLR4 inflammatory responses. We then performed extended incubation of cells with physiological concentrations of small alpha-synuclein oligomers observing the development of a TLR4-dependent sensitized inflammatory response with time, including TNF-α production. ROS and cell death in primary neuronal cultures were significantly reduced by TLR4 antagonists revealing that an indirect inflammatory mechanism involving cytokines produced by glial cells makes a major contribution to neuronal death. Prolonged exposure to low levels of alpha-synuclein oligomers sensitizes TLR4 responsiveness in astrocytes and microglial, explaining how they become pro-inflammatory, and may be an early causative event in PD

SYNTHESIS AND CHARACTERIZATION OF POLYMERIC MICELLE DELIVERY SYSTEM AS A DRUG AND

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. There is currently no effective therapeutic for the treatment of TBI. Primary injury, from the initial injury, causes contusions and hemorrhaging at the site of impact. Diffuse damage is caused throughout the brain from the impact; this includes axonal injury, hypoxic brain damage, brain swelling and vascular injury. Brain damage continues as the secondary injury; which is characterized by hypoxia, hypotension, amino acid excitotoxicity and ionic imbalance. All of these conditions cause additional cell death and damage. Inflammation, brought about by reduced cyclic AMP levels is also seen post-injury. After injury, the glial scar and myelin produce neurite growth inhibitory molecules. There are several different types of myelin associated inhibitors expressed by oligodendrocytes; these interact with multiple types of neuron surface receptors triggering the RhoA cascade, which inhibits actin polymerization and neurite outgrowth. Chondroitin sulfate proteoglycans (CSPG) expressed on astrocytes also inhibits growth through the same RhoA pathway. Several strategies have elected to knockdown the RhoA gene or other genes involved in the growth inhibition pathway. The objective of this work is to develop novel neuron-specific nanotherapeutics for combinatorial therapy of drug and small interfering RNA (siRNA) targeting both extrinsic and intrinsic barriers to neuroplasticity. This neuron-specific polymeric micelle nanotherapeutics will be designed as follows: First, a cell-type specific targeting moiety such as an antibody can be conjugated to the polymeric micelle nanoparticle surface to deliver nanotherapeutics specifically to neurons. Second, RhoA siRNA, can be targeted to common intracellular signal transduction pathways for inhibitory molecules such as myelin and CSPGs. Third, a hydrophobic drug, a phosphodiesterase 4 inhibitor (rolipram) will be incorporated in the PgP micelle to increase intrinsic neuronal growth capacity by preventing injury-induced reductions in cAMP levels. To achieve this goal, we synthesized amphiphilic block copolymers, poly (lactide-co-glycolide)-graft-polyethylenimine (PLGA-g-PEI: PgP) using PLGA as a hydrophobic core forming block and PEI as a hydrophilic shell forming block and characterized the physico-chemical properties of the PgP micelle as a delivery carrier for combinatorial therapy of nucleic acid and drug. We demonstrated that the PgP micelle is a promising nucleic acid delivery carrier using phMGFP plasmid as a reporter gene in C6 (glioma) cells and primary chick forebrain neurons (CFN) cells in 10% serum containing media in vitro. We also studied incorporating rolipram in the PgP micelle and successfully conjugated an antibody (mouse IgG) on the surface of PgP. Currently, we are evaluating PgP as a siRNA delivery carrier to primary CFN cells and preparing PgP-mNgR1 using NgR1 monoclonal antibody and evaluating the feasibility of PgP-mNgR1 as a neuron-specific nucleic acid carrier for targeting neuron cells in a rat cortical neuron /astrocyte co-culture system. In the future, we will study rolipram-loaded PgP-Ab as a nucleic acid/drug carrier using RhoA siRNA in hypoxic conditions as a TBI model in vitro and a rat traumatic brain injury model in vivo