Co-cultures Of Astrocytes Research Articles

Abstract TMP74: 3-HKA Promotes the Vascular Remodeling after Stroke by Modulating the Activation of A1/A2 Reactive Astrocytes

Background: Ischemic stroke is the most common cerebrovascular disease worldwide and the leading cause of permanent disability, which imposes a heavy burden on society and families. Currently, there are still limited treatments for improving long-term recovery after cerebral infarction. Recent studies have found that the development and prognosis after stroke are closely related to abnormal tryptophan metabolism. In this study, we aimed to identify tryptophan metabolites with ischemic protective effects in stroke patients and animal models and determine their mechanisms of action. Methods: Metabolomics analysis using liquid chromatography-mass spectrometry was performed to identify differentially bioactive metabolites in plasma samples from 46 acute ischemic stroke (AIS) patients and 35 healthy controls and validated in experimental animals, identifying decreased levels of 3-hydroxy-kynurenamine (3-HKA) after stroke. We then examined the role of 3-HKA in stroke mice and performed proteomic analysis to elucidate the molecular mechanisms of 3-HKA. Results: Supplementation of 3-HKA improves long-term sensory and motor recovery in ischemic stroke and increases ipsilateral cerebral blood flow. 3-HKA can also promote angiogenesis, stimulate the formation of functional blood vessels, and repair the blood-brain barrier, thereby enhancing post-stroke remodeling. Meanwhile, 3-HKA inhibits the activation of neurotoxic (A1-like) astrocytes but promotes the polarization of neuroprotective (A2-like) astrocytes. Mechanistically, 3-HKA inhibited the activation of AIM2 inflammasomes, and AIM2 expression was increased mainly in astrocytes at 7 and 14 days after stroke, suggesting that 3-HKA may regulate astrocyte-mediated vascular remodeling. Consistently, in primary mouse brain microvascular endothelial cells and astrocyte co-culture, 3-HKA promoted angiogenesis after oxygen-glucose deprivation (OGD). In astrocytes, the effects of 3-HKA on enhancing vascular remodeling in vitro and in vivo were abolished after lentiviral or AAV overexpression of AIM2. Overexpression of AIM2 in astrocytes by lentivirus or AAV counteracted the effect of 3-HKA on augmenting vascular remodeling in vitro and in vivo. Conclusions: Our results suggest that tryptophan metabolism is disturbed after stroke, with decreased 3-HKA levels. Supplementation of 3-HKA may foster vascular remodeling by regulating the activation of A1/A2 astrocytes after stroke, thereby improving long-term neurological recovery.

3-HKA Promotes Vascular Remodeling After Stroke by Modulating the Activation of A1/A2 Reactive Astrocytes.

Ischemic stroke is the most common cerebrovascular disease and the leading cause of permanent disability worldwide. Recent studies have shown that stroke development and prognosis are closely related to abnormal tryptophan metabolism. Here, significant downregulation of 3-hydroxy-kynurenamine (3-HKA) in stroke patients and animal models is identified. Supplementation with 3-HKA improved long-term neurological recovery, reduced infarct volume, and increased ipsilateral cerebral blood flow after distal middle cerebral artery occlusion (MCAO). 3-HKA promoted angiogenesis, functional blood vessel formation, and blood-brain barrier (BBB) repair. Moreover, 3-HKA inhibited A1-like (neurotoxic) astrocyte activation but promoted A2-like (neuroprotective) astrocyte polarization. Proteomic analysis revealed that 3-HKA inhibited AIM2 inflammasome activation after stroke, and co-labeling studies indicated that AIM2 expression typically increased in astrocytes at 7 and 14 days after stroke. Consistently, in co-cultures of primary mouse brain microvascular endothelial cells and astrocytes, 3-HKA promoted angiogenesis after oxygen-glucose deprivation (OGD). AIM2 overexpression in astrocytes abrogated 3-HKA-driven vascular remodeling in vitro and in vivo, suggesting that 3-HKA may regulate astrocyte-mediated vascular remodeling by impeding AIM2 inflammasome activation. In conclusion, 3-HKA may promote post-stroke vascular remodeling by regulating A1/A2 astrocyte activation, thereby improving long-term neurological recovery, suggesting that supplementation with 3-HKA may be an efficient therapy for stroke.

Non-Invasive Measurement of Elasticity in Glioblastoma Multiforme Validates Decreased TMZ Sensitivity in Astrocyte Co-Culture

Non-Invasive Measurement of Elasticity in Glioblastoma Multiforme Validates Decreased TMZ Sensitivity in Astrocyte Co-Culture

Down-regulation of platelet-derived growth factor receptor β in pericytes increases blood-brain barrier permeability and significantly enhances α-synuclein in a Parkinson's Disease 3D cell model in vitro under hyperglycemic condition.

Parkinson's Disease (PD) often presents with a compromised blood-brain barrier (BBB), which hyperglycemia may exacerbate. Pericytes, a key cell for BBB integrity, are potential therapeutic targets for neurodegenerative disorders. Few studies have developed 3D PD cell models incorporating neurovascular units (NVU) through the co-culture of human endothelial, pericytes, astrocytes, and SH-SY5Y cells to evaluate BBB impairment and the role of pericytes under hyperglycemic condition. A 3D PD like cell model was developed using 6-OHDA-affected SH-SY5Y cells, combined with endothelial cells, pericytes, and astrocytes through the Real Architecture for Tissue (RAFT) 3D co-culture system. PD incorporating reduced (30 % and 89 %) PDGFRβ NVU (RPN) with or without hyperglycemic model (HM) were also established. BBB permeability to sodium fluorescein was assessed, and BBB impairment was evaluated using BBB-associated proteins (ZO-1, CD54, CD144), cell-specific proteins (CD31, GFAP, PDGFRβ, CD13), tyrosine hydroxylase (TH), α-synuclein, oligomeric α-synuclein, and α-synuclein (ser9). PD 3D cell models incorporating RPN with or without hyperglycemia were successfully established in vitro. Graduately increased BBB impairment was observed in PD, PD with RPN, and PD with RPN combined with HM, indicated by decreased BBB-associated and cell-specific proteins, reduced TH, and increased α-synuclein, oligomeric α-synuclein, and α-synuclein (ser9) compared to the NVU model. Reduced pericyte PDGFRβ could increase BBB permeability, accelerate PD progression, and exacerbate under hyperglycemic condition.

Na+-K+-ATPase/GLT-1 interaction participates in EGCG protection against cerebral ischemia-reperfusion injury in rats.

In China, stroke is the primary cause of adult death and disability. Because of the increased rate of blood vessel reperfusion, it is important to prevent cerebral ischemia-reperfusion injury, in which glutamate (Glu) excitotoxicity plays a critical role. The most important Glu transporter, GLT-1, is essential for the regulation of Glu, which is dependent on Na+-K+-ATPase (NKA)-induced ion concentration gradient differences. EGCG, a substance found in tea polyphenols, can reduce infarct areas in ischemia-reperfusion models, reduce stroke incidence, and prolong life in which NKA is involved. In this study, we investigated the potential of EGCG in protecting against cerebral ischemia-reperfusion injury by regulating the interaction between NKA and GLT-1. This study was designed to investigate the protective effects of EGCG against cerebral ischemia-reperfusion injury by modulating the interaction between NKA and GLT-1, utilizing both the rat middle cerebral artery occlusion/reperfusion (MCAO/R) model and the oxygen-glucose deprivation/reoxygenation (OGD/R) model in co-cultures of rat hippocampal neurons and astrocytes. The neuronal survival rate was assessed using CCK8, and the cerebral infarction area and neurological function were determined by TTC staining and neurological deficit scores. NKA activity was measured using an inorganic phosphorous detection method, and NKA and GLT-1 expression was detected using western blotting. The interaction between NKAα2 and GLT-1 was identified by co-immunoprecipitation (CoIP) assay, laser confocal microscopy, and Imaris 3D confocal rendering technology. An adenovirus vector with overexpression of NKAα2 was constructed, packaged, and injected into the rat lateral ventricle. Neurological function and the cerebral infarction area were identified, and the interaction between NKAα2 and GLT-1 was identified using CoIP assay. EGCG reduced the infarction area and neurological deficit scores, restored NKA activity, alleviated the decrease in membrane NKAα2 and GLT-1 expression, and relieved the uncoupling of NKAα2 and GLT-1 in the hippocampal CA1 after rat MCAO/R injury. By promoting the coupling of NKAα2 and GLT-1 in rat MCAO/R models, overexpression of NKAα2 reduced the cerebral infarction area and neurological impairment scores. EGCG improved cerebral ischemia-reperfusion injury by restoring NKA activity and increasing membrane GLT-1 expression due to NKA-GLT-1 interaction. For the first time, our findings demonstrate the critical role that NKA and GLT-1 colocalization plays in cerebral ischemia-reperfusion damage. Our findings provide new strategic directions for the pathogenesis and prevention of thrombolytic injury in the clinical treatment of stroke, while also serving as a basis for further development and utilization of EGCG.

Electrochemical imaging of neurotransmitter release with fast-scan voltammetric ion conductance microscopy.

Understanding the dynamic spatial and temporal release of neurotransmitters can help resolve long-standing questions related to chemical modulation of neurological circuits. Dopamine modulates function in a range of physiological processes and is key to transmission in addiction and neurological disorders. Studies at subcellular scales promise to help develop a broader understanding of dopamine release, diffusion, and receptor activation and how these processes lead to functional outcomes. Electrochemical measurements of dopamine release at individual cells have proven especially informative. We describe incorporation of fast-scan cyclic voltammetry for detection of dopamine release with subcellular spatial resolution and millisecond time resolution. The platform is benchmarked with standard redox probes and then applied to imaging stimulated release from subcellular locations of a coculture of dopaminergic neurons and astrocytes. Voltammetry reveals heterogeneity in release based on time, location, and neuron identity. We believe that this platform ultimately offers a window to understanding neurotransmission in pathophysiological models of disease where cell-cell communication is key.

Application of an in vitro neuroinflammation model to evaluate the efficacy of magnesium-lithium alloys.

Mg-Li alloys can be promising candidates as bioresorbable Li-releasing implants for bipolar disorder and other neurodegenerative disorders. In order to compare the therapeutic efficacy of conventional Li salts and Li delivered through Mg-Li alloy extracts, we tested an in vitro model based on the neuroinflammation hypothesis of mood disorders (peripheral inflammation inducing neuroinflammation) wherein, a coculture of microglia and astrocytes was treated with conditioned medium from pro-inflammatory macrophages. Two alloys, Mg-1.6Li and Mg-9.5Li, were tested in the form of material extracts and well-known outcomes of Li treatment such as GSK3β phosphorylation (indirect flow cytometry) and influence on inflammation-related gene expression (qPCR) were compared against Li salts. This is the first study demonstrating that Li can increase the phosphorylation of GSK3β in glial cells in the presence of excess Mg. Furthermore, Mg-Li alloys were more effective than Li salts in downregulating IL6 and upregulating the neurotrophin GDNF. Mg had no antagonistic effects toward Li-driven downregulation of astrogliosis markers. Overall, the results provide evidence to support further studies employing Mg-Li alloys for neurological applications.

APPLICATION OF IN VITRO MODELS: INCORPORATING BLOOD BRAIN BARRIER MODELS WITH GLIOMA, AND THE USE OF MICROflUIDICS

Abstract AIMS The blood brain barrier (BBB) presents one of the main obstacles for the development of novel glioblastoma treatments. The various transporters and enzymes within the BBB reduce the disposition of novel therapies into the brain. There is a need for an in vitro BBB-glioma model with proven efficacy of predicting CNS delivery at earlier stages in the drug discovery pipeline. METHOD A BBB-glioma lab-on-a-chip model could enable rapid identification of novel therapies that can cross the BBB at earlier stages of the drug development process, before going to clinical trials. The application of microfluidic technology allows for the detection of soluble tissue-derived factors and the assessment of the levels of soluble markers released from malignant tissue. Adding ‘flow’ to the model, is more reflective of the dynamic flow present at the BBB. RESULTS Validation studies of the current BBB model and converting this model over to a lab-on-a-chip model have shown the presence of an endothelial cell barrier and the tight junction proteins Zo-1 and Claudin-1, through western blotting, immunocytochemistry, and permeability experiments using varying molecular weights of FitC-dextran. These validation studies have been completed using brain endothelial monolayers on polycarbonate transwell inserts, with the immortalised cell line HCMEC/d3, and the short-term culture cells HBMEC. Also, validation studies using a co-culture of endothelial cells, astrocytes and pericytes have begun, using the same techniques to show a barrier has formed, on both the current BBB model and the lab-on-a-chip model. CONCLUSION The aim of this study is to combine the use of an all-human BBB-glioma model with lab-on-a-chip technology to produce a high throughput model to test novel therapies at an earlier stage. The lab-on-a-chip model will be validated for the purpose of measuring permeability and drug response and to investigate the expression of novel targets verses expression in conventional models.

Astrocytes Can Be Key Players Against Cerebral Leishmaniasis: InVitro Co-Culture Model for the Assessment of Infection.

Leishmaniasis is a neglected tropical disease, caused by protozoan parasites of Leishmania (L.), and is transmitted by bite of phlebotomine sandflies. There are several studies on central nervous system infection to indicate that Leishmania can cross the blood-brain barrier, resulting in neurological manifestations, known as "cerebral leishmaniasis." This study highlighted the notions: (i) polarisation of bone marrow-derived macrophages (BMDM) incubated following stimulation with lipopolysaccharide (LPS) or soluble Leishmania antigen (SLA), (ii) quantification of parasites within co-culture of Leishmania-infected macrophages, and astrocytes, and (iii) effect of interferon-gamma (IFN-γ) on the infection rate of co-culture populations. Accordingly, 83% of overall macrophage population was identified on day 7 for CD11b and F4/80 macrophage markers. Flow cytometry analysis revealed significant increases in CD11b and F4/80 surface markers in LPS and SLA-stimulated BMDMs at 24 h, compared to untreated cells. TNF-α levels increased significantly in both LPS and SLA-treated BMDMs after 48 h. Additionally, SLA treatment induced a more elongated, spindle-like shape in the cells, indicative of M2 macrophage polarisation over the M1 phenotype. When non-infected astrocytes with/without stimulation with IFN-γ before co-culture, gp63 FITC-labelled parasite populations (%) in co-culture decreased to 25% at 72 h, thus indicating a lower infection rate in a time-dependent manner. IFN-γ and IL-6 levels significantly increased to 71.66 ± 3.51 and 184 ± 14.42 pg/mL, resulting in the inflammatory response in the co-culture system at 48 h (p ≤ 0.0001), when compared to the control (30 ± 2.52 pg/mL for IFN-γ and 8.66 ± 2.37 pg/mL for IL-6) at 0 h of the incubation. It is the first study to emphasize the communication between Leishmania-infected macrophages and astrocytes regarding Leishmania parasite load. The results suggest that astrocytes can lead to the reduction in Leishmania parasites, thereby controlling the incidence of cerebral leishmaniasis.

Aberrant neurodevelopment in human iPS cell-derived models of Alexander disease.

Alexander disease (AxD) is a rare and severe neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP). While the exact disease mechanism remains unknown, previous studies suggest that mutant GFAP influences many cellular processes, including cytoskeleton stability, mechanosensing, metabolism, and proteasome function. While most studies have primarily focused on GFAP-expressing astrocytes, GFAP is also expressed by radial glia and neural progenitor cells, prompting questions about the impact of GFAP mutations on central nervous system (CNS) development. In this study, we observed impaired differentiation of astrocytes and neurons in co-cultures of astrocytes and neurons, as well as in neural organoids, both generated from AxD patient-derived induced pluripotent stem (iPS) cells with a GFAPR239C mutation. Leveraging single-cell RNA sequencing (scRNA-seq), we identified distinct cell populations and transcriptomic differences between the mutant GFAP cultures and a corrected isogenic control. These findings were supported by results obtained with immunocytochemistry and proteomics. In co-cultures, the GFAPR239C mutation resulted in an increased abundance of immature cells, while in unguided neural organoids and cortical organoids, we observed altered lineage commitment and reduced abundance of astrocytes. Gene expression analysis revealed increased stress susceptibility, cytoskeletal abnormalities, and altered extracellular matrix and cell-cell communication patterns in the AxD cultures, which also exhibited higher cell death after stress. Overall, our results point to altered cell differentiation in AxD patient-derived iPS-cell models, opening new avenues for AxD research.

Induction of astrocyte reactivity promotes neurodegeneration in human pluripotent stem cell models

Reactive astrocytes are known to exert detrimental effects upon neurons in several neurodegenerative diseases, yet our understanding of how astrocytes promote neurotoxicity remains incomplete, especially in human systems. In this study, we leveraged human pluripotent stem cell (hPSC) models to examine how reactivity alters astrocyte function and mediates neurodegeneration. hPSC-derived astrocytes were induced to a reactive phenotype, at which point they exhibited a hypertrophic profile and increased complement C3 expression. Functionally, reactive astrocytes displayed decreased intracellular calcium, elevated phagocytic capacity, and decreased contribution to the blood-brain barrier. Subsequently, co-culture of reactive astrocytes with a variety of neuronal cell types promoted morphological and functional alterations. Furthermore, when reactivity was induced in astrocytes from patient-specific hPSCs (glaucoma, Alzheimer's disease, and amyotrophic lateral sclerosis), the reactive state exacerbated astrocytic disease-associated phenotypes. These results demonstrate how reactive astrocytes modulate neurodegeneration, significantly contributing to our understanding of a role for reactive astrocytes in neurodegenerative diseases.

Restoring hippocampal glucose metabolism rescues cognition across Alzheimer's disease pathologies.

Impaired cerebral glucose metabolism is a pathologic feature of Alzheimer Disease (AD), and recent proteomic studies highlight a disruption of glial carbohydrate metabolism with disease progression. Here, we report that inhibition of indoleamine-2,3-dioxygenase 1 (IDO1), which metabolizes tryptophan to kynurenine (KYN) in the first step of the kynurenine pathway, rescues hippocampal memory function and plasticity in preclinical models of amyloid and tau pathology by restoring astrocytic metabolic support of neurons. Activation of IDO1 in astrocytes by amyloid-beta 42 and tau oligomers, two major pathological effectors in AD, increases KYN and suppresses glycolysis in an AhR-dependent manner. Conversely, pharmacological IDO1 inhibition restores glycolysis and lactate production. In amyloid-producing APP Swe -PS1 ΔE9 and 5XFAD mice and in tau-producing P301S mice, IDO1 inhibition restores spatial memory and improves hippocampal glucose metabolism by metabolomic and MALDI-MS analyses. IDO1 blockade also rescues hippocampal long-term potentiation (LTP) in a monocarboxylate transporter (MCT)-dependent manner, suggesting that IDO1 activity disrupts astrocytic metabolic support of neurons. Indeed, in vitro mass-labeling of human astrocytes demonstrates that IDO1 regulates astrocyte generation of lactate that is then taken up by human neurons. In co-cultures of astrocytes and neurons derived from AD subjects, deficient astrocyte lactate transfer to neurons was corrected by IDO1 inhibition, resulting in improved neuronal glucose metabolism. Thus, IDO1 activity disrupts astrocytic metabolic support of neurons across both amyloid and tau pathologies and in a model of AD iPSC-derived neurons. These findings also suggest that IDO1 inhibitors developed for adjunctive therapy in cancer could be repurposed for treatment of amyloid- and tau-mediated neurodegenerative diseases.

Anti-Inflammatory Action of Resveratrol in the Central Nervous System in Relation to Glucose Concentration-An In Vitro Study on a Blood-Brain Barrier Model.

Unbalanced blood glucose levels may cause inflammation within the central nervous system (CNS). This effect can be reversed by the action of a natural neuroprotective compound, resveratrol (RSV). The study aimed to investigate the anti-inflammatory effect of RSV on astrocyte cytokine profiles within an in vitro model of the blood-brain barrier (BBB) under varying glucose concentrations (2.2, 5.0, and 25.0 mmol/L), corresponding to hypo-, normo-, and hyperglycemia. The model included co-cultures of astrocytes (brain compartment, BC) and endothelial cells (microvascular compartment, MC), separated by 0.4 µm wide pores. Subsequent exposure to 0.2 μM LPS in the brain compartment (BC) and 50 μM RSV in the microvascular compartment (MC) of each well was carried out. Cytokine levels (IL-1 α, IL-1 β, IL-2, IL-4, IL-6, IL-8) in the BC were assessed using a Multi-Analyte ELISArray Kit before and after the addition of LPS and RSV. Statistical analysis was performed to determine significance levels. The results demonstrated that RSV reduced the concentration of all studied cytokines in the BC, regardless of glucose levels, with the most substantial decrease observed under normoglycemic conditions. Additionally, the concentration of RSV in the BC was highest under normoglycemic conditions compared to hypo- and hyperglycemia. These findings confirm that administration of RSV in the MC exerts anti-inflammatory effects within the BC, particularly under normoglycemia-simulating conditions. Further in vivo studies, including animal and human research, are warranted to elucidate the bioavailability of RSV within the central nervous system (CNS).

Porous Polymeric Nanofilms for Recreating the Basement Membrane in an Endothelial Barrier-on-Chip.

Organs-on-chips (OoCs) support an organotypic human cell culture in vitro. Precise representation of basement membranes (BMs) is critical for mimicking physiological functions of tissue interfaces. Artificial membranes in polyester (PES) and polycarbonate (PC) commonly used in in vitro models and OoCs do not replicate the characteristics of the natural BMs, such as submicrometric thickness, selective permeability, and elasticity. This study introduces porous poly(d,l-lactic acid) (PDLLA) nanofilms for replicating BMs in in vitro models and demonstrates their integration into microfluidic chips. Using roll-to-roll gravure coating and polymer phase separation, we fabricated transparent ∼200 nm thick PDLLA films. These nanofilms are 60 times thinner and 27 times more elastic than PES membranes and show uniformly distributed pores of controlled diameter (0.4 to 1.6 μm), which favor cell compartmentalization and exchange of large water-soluble molecules. Human umbilical vein endothelial cells (HUVECs) on PDLLA nanofilms stretched across microchannels exhibited 97% viability, enhanced adhesion, and a higher proliferation rate compared to their performance on PES membranes and glass substrates. After 5 days of culture, HUVECs formed a functional barrier on suspended PDLLA nanofilms, confirmed by a more than 10-fold increase in transendothelial electrical resistance and blocked 150 kDa dextran diffusion. When integrated between two microfluidic channels and exposed to physiological shear stress, despite their ultrathin thickness, PDLLA nanofilms upheld their integrity and efficiently maintained separation of the channels. The successful formation of an adherent endothelium and the coculture of HUVECs and human astrocytes on either side of the suspended nanofilm validate it as an artificial BM for OoCs. Its submicrometric thickness guarantees intimate contact, a key feature to mimic the blood-brain barrier and to study paracrine signaling between the two cell types. In summary, porous PDLLA nanofilms hold the potential for improving the accuracy and physiological relevance of the OoC as in vitro models and drug discovery tools.

RAGE induces physiological activation of NADPH oxidase in neurons and astrocytes and neuroprotection.

The transmembrane receptor for advanced glycation end products (RAGE) is a signaling receptor for many damage- and pathogen-associated molecules. Activation of RAGE is associated with inflammation and an increase in reactive oxygen species (ROS) production. Although several sources of ROS have been previously suggested, how RAGE induces ROS production is still unclear, considering the multiple targets of pathogen-associated molecules. Here, using acute brain slices and primary co-culture of cortical neurons and astrocytes, we investigated the effects of a range of synthetic peptides corresponding to the fragments of the RAGE V-domain on redoxsignaling. We found that the synthetic fragment (60-76) of the RAGE V-domain induces activation of ROS production in astrocytes and neurons from the primary co-culture and acute brain slices. This effect occurred through activation of RAGE and could be blocked by a RAGE inhibitor. Activation of RAGE by the synthetic fragment stimulates ROS production in NADPH oxidase (NOX). This RAGE-induced NOX activation produced only minor decreases in glutathione levels and increased the rate of lipid peroxidation, although it also reduced basal and β-amyloid induced cell death in neurons and astrocytes. Thus, specific activation of RAGE induces redox signaling through NOX, which can be a part of a cell protective mechanism.

ATH-1105, a small-molecule positive modulator of the neurotrophic HGF system, is neuroprotective, preserves neuromotor function, and extends survival in preclinical models of ALS.

Amyotrophic lateral sclerosis (ALS), a progressive and fatal neurodegenerative disorder, primarily affects the motor neurons of the brain and spinal cord. Like other neurodegenerative conditions, ongoing pathological processes such as increased inflammation, excitotoxicity, and protein accumulation contribute to neuronal death. Hepatocyte growth factor (HGF) signaling through the MET receptor promotes pro-survival, anti-apoptotic, and anti-inflammatory effects in multiple cell types, including the neurons and support cells of the nervous system. This pleiotropic system is therefore a potential therapeutic target for treatment of neurodegenerative disorders such as ALS. Here, we test the effects of ATH-1105, a small-molecule positive modulator of the HGF signaling system, in preclinical models of ALS. In vitro, the impact of ATH-1105 on HGF-mediated signaling was assessed via phosphorylation assays for MET, extracellular signal-regulated kinase (ERK), and protein kinase B (AKT). Neuroprotective effects of ATH-1105 were evaluated in rat primary neuron models including spinal motor neurons, motor neuron-astrocyte cocultures, and motor neuron-human muscle cocultures. The anti-inflammatory effects of ATH-1105 were evaluated in microglia- and macrophage-like cell systems exposed to lipopolysaccharide (LPS). In vivo, the impact of daily oral treatment with ATH-1105 was evaluated in Prp-TDP43A315T hemizygous transgenic ALS mice. In vitro, ATH-1105 augmented phosphorylation of MET, ERK, and AKT. ATH-1105 attenuated glutamate-mediated excitotoxicity in primary motor neurons and motor neuron- astrocyte cocultures, and had protective effects on motor neurons and neuromuscular junctions in motor neuron-muscle cocultures. ATH-1105 mitigated LPS-induced inflammation in microglia- and macrophage-like cell systems. In vivo, ATH-1105 treatment resulted in improved motor and nerve function, sciatic nerve axon and myelin integrity, and survival in ALS mice. Treatment with ATH-1105 also led to reductions in levels of plasma biomarkers of inflammation and neurodegeneration, along with decreased pathological protein accumulation (phospho-TDP-43) in the sciatic nerve. Additionally, both early intervention (treatment initiation at 1 month of age) and delayed intervention (treatment initiation at 2 months of age) with ATH-1105 produced benefits in this preclinical model of ALS. The consistent neuroprotective and anti-inflammatory effects demonstrated by ATH-1105 preclinically provide a compelling rationale for therapeutic interventions that leverage the positive modulation of the HGF pathway as a treatment for ALS.

GLT-1a glutamate transporter nanocluster localization is associated with astrocytic actin and neuronal Kv2 clusters at sites of neuron-astrocyte contact.

Introduction: Astrocytic GLT-1 glutamate transporters ensure the fidelity of glutamic neurotransmission by spatially and temporally limiting glutamate signals. The ability to limit neuronal hyperactivity relies on the localization and diffusion of GLT-1 on the astrocytic surface, however, little is known about the underlying mechanisms. We show that two isoforms of GLT-1, GLT-1a and GLT-1b, form nanoclusters on the surface of transfected astrocytes and HEK-293 cells. Methods: We used both fixed and live cell super-resolution imaging of fluorescent protein and epitope tagged proteins in co-cultures of rat astrocytes and neurons. Immunofluorescence techniques were also used. GLT1 diffusion was assessed via single particle tracking and fluorescence recovery after photobleach (FRAP). Results: We found GLT-1a, but not GLT-1b, nanoclusters concentrated adjacent to actin filaments which was maintained after addition of glutamate. GLT-1a nanocluster concentration near actin filaments was prevented by expression of a cytosolic GLT-1a C-terminus, suggesting the C-terminus is involved in the localization adjacent to cortical actin. Using super-resolution imaging, we show that astrocytic GLT-1a and actin co-localize in net-like structures around neuronal Kv2.1 clusters at points of neuron/astrocyte contact. Conclusion: Overall, these data describe a novel relationship between GLT-1a and cortical actin filaments, which localizes GLT-1a near neuronal structures responsive to ischemic insult.

Excitatory to inhibitory synaptic ratios are unchanged at presymptomatic stages in multiple models of ALS.

Hyperexcitability of motor neurons and spinal cord motor circuitry has been widely reported in the early stages of Amyotrophic Lateral Sclerosis (ALS). Changes in the relative amount of excitatory to inhibitory inputs onto a neuron (E:I synaptic ratio), possibly through a developmental shift in synapse formation in favour of excitatory transmission, could underlie pathological hyperexcitability. Given that astrocytes play a major role in early synaptogenesis and are implicated in ALS pathogenesis, their potential contribution to disease mechanisms involving synaptic imbalances and subsequent hyperexcitability is also of great interest. In order to assess E:I ratios in ALS, we utilised a novel primary spinal neuron / astrocyte co-culture system, derived from neonatal mice, in which synapses are formed in vitro. Using multiple ALS mouse models we found that no combination of astrocyte or neuron genotype produced alterations in E:I synaptic ratios assessed using pre- and post-synaptic anatomical markers. Similarly, we observed that ephrin-B1, a major contact-dependent astrocytic synaptogenic protein, was not differentially expressed by ALS primary astrocytes. Further to this, analysis of E:I ratios across the entire grey matter of the lumbar spinal cord in young (post-natal day 16-19) ALS mice revealed no differences versus controls. Finally, analysis in co-cultures of human iPSC-derived motor neurons and astrocytes harbouring the pathogenic C9orf72 hexanucleotide repeat expansion showed no evidence of a bias toward excitatory versus inhibitory synapse formation. We therefore conclude, utilising multiple ALS models, that we do not observe significant changes in the relative abundance of excitatory versus inhibitory synapses as would be expected if imbalances in synaptic inputs contribute to early hyperexcitability.

High levels of FAD autofluorescence indicate pathology preceding cell death

Flavin adenine dinucleotide (FAD) autofluorescence from cells reports on the enzymatic activity which involves FAD as a cofactor. Most of the cellular FAD fluorescence comes from complex II of the electron transport chain in mitochondria and can be assessed with inhibitor analysis. The intensity of FAD autofluorescence is not homogeneous and vary between cells in tissue and in cell culture types. Using primary co-culture of neurons and astrocytes, and human skin fibroblasts we have found that very high FAD autofluorescence is a result of an overactivation of the mitochondrial complex II from ETC and from the activity of monoamine oxidases. Cells with high FAD autofluorescence were mostly intact and were not co-labelled with indicators for necrosis or apoptosis. However, cells with high FAD fluorescence showed activation of apoptosis and necrosis within 24 h after initial measurements. Thus, high level of FAD autofluorescence is an indicator of cell pathology and reveals an upcoming apoptosis and necrosis.

Trilobatin attenuates cerebral ischaemia/reperfusion-induced blood-brain barrier dysfunction by targeting matrix metalloproteinase 9: The legend of a food additive.

Blood-brain barrier (BBB) breakdown is one of the crucial pathological changes of cerebral ischaemia-reperfusion (I/R) injury. Trilobatin (TLB), a naturally occurring food additive, exerts neuroprotective effects against cerebral I/R injury as demonstrated in our previous study. This study was designed to investigate the effect of TLB on BBB disruption after cerebral I/R injury. Rats with focal cerebral ischaemia caused by transient middle cerebral artery occlusion were studied along with brain microvascular endothelial cells and human astrocytes to mimic BBB injury caused by oxygen and glucose deprivation/reoxygenation (OGD/R). The results showed that TLB effectively maintained BBB integrity and inhibited neuronal loss following cerebral I/R challenge. Furthermore, TLB increased tight junction proteins including ZO-1, Occludin and Claudin 5, and decreased the levels of apolipoprotein E (APOE) 4, cyclophilin A (CypA) and phosphorylated nuclear factor kappa B (NF-κB), thereby reducing proinflammatory cytokines. TLB also decreased the Bax/Bcl-2 ratio and cleaved-caspase 3 levels along with a reduced number of apoptotic neurons. Molecular docking and transcriptomics predicted MMP9 as a prominent gene evoked by TLB treatment. The protective effects of TLB on cerebral I/R-induced BBB breakdown was largely abolished by overexpression of MMP9, and the beneficial effects of TLB on OGD/R-induced loss of BBB integrity in human brain microvascular endothelial cells and astrocyte co-cultures was markedly reinforced by knockdown of MMP9. Our findings reveal a novel property of TLB: preventing BBB disruption following cerebral I/R via targeting MMP9 and inhibiting APOE4/CypA/NF-κB axis.