Marta Sidoryk-Wegrzynowicz

SyM-BBB: A Microfluidic Blood Brain Barrier Model

Current techniques for mimicking the Blood-Brain Barrier (BBB) largely use incubation chambers (Transwell) separated with a filter and matrix coating to represent and to study barrier permeability. These devices have several critical shortcomings: (a) they do not reproduce critical microenvironmental parameters, primarily anatomical size or hemodynamic shear stress, (b) they often do not provide real-time visualization capability, and (c) they require a large amount of consumables. To overcome these limitations, we have developed a microfluidics based Synthetic Microvasculature model of the Blood-Brain Barrier (SyM-BBB). The SyM-BBB platform is comprised of a plastic, disposable and optically clear microfluidic chip with a microcirculation sized two-compartment chamber. The chamber is designed in such a way as to permit the realization of side-by-side apical and basolateral compartments, thereby simplifying fabrication and facilitating integration with standard instrumentation. The individually addressable apical side is seeded with endothelial cells and the basolateral side can support neuronal cells or conditioned media. In the present study, an immortalized Rat Brain Endothelial cell line (RBE4) was cultured in SyM-BBB with a perfusate of Astrocyte Conditioned Media (ACM). Biochemical analysis showed upregulation of tight junction molecules while permeation studies showed an intact BBB. Finally, transporter assay was successfully demonstrated in SyM-BBB indicating a functional model.

Role of Astrocytes in Brain Function and Disease

Astrocytes assume multiple roles in maintaining an optimally suited milieu for neuronal function. Select astrocytic functions include the maintenance of redox potential, the production of trophic factors, the regulation of neurotransmitter and ion concentrations, and the removal of toxins and debris from the cerebrospinal fluid (CSF). Impairments in these and other functions, as well as physiological reactions of astrocytes to injury, can trigger or exacerbate neuronal dysfunction. This review addresses select metabolic interactions between neurons and astrocytes and emphasizes the role of astrocytes in mediating and amplifying the progression of several neurodegenerative disorders, such as Parkinson’s disease (PD), hepatic encephalopathy (HE), hyperammonemia (HA), Alzheimer’s disease (AD), and ischemia.

Roles of glutamine in neurotransmission

Glutamine (Gln) is found abundantly in the central nervous system (CNS) where it participates in a variety of metabolic pathways. Its major role in the brain is that of a precursor of the neurotransmitter amino acids: the excitatory amino acids, glutamate (Glu) and aspartate (Asp), and the inhibitory amino acid, γ-amino butyric acid (GABA). The precursor-product relationship between Gln and Glu/GABA in the brain relates to the intercellular compartmentalization of the Gln/Glu(GABA) cycle (GGC). Gln is synthesized from Glu and ammonia in astrocytes, in a reaction catalyzed by Gln synthetase (GS), which, in the CNS, is almost exclusively located in astrocytes (Martinez-Hernandez et al., 1977). Newly synthesized Gln is transferred to neurons and hydrolyzed by phosphate-activated glutaminase (PAG) to give rise to Glu, a portion of which may be decarboxylated to GABA or transaminated to Asp. There is a rich body of evidence which indicates that a significant proportion of the Glu, Asp and GABA derived from Gln feed the synaptic, neurotransmitter pools of the amino acids. Depolarization-induced-, calcium- and PAG activity-dependent releases of Gln-derived Glu, GABA and Asp have been observed in CNS preparations in vitro and in the brain in situ. Immunocytochemical studies in brain slices have documented Gln transfer from astrocytes to neurons as well as the location of Gln-derived Glu, GABA and Asp in the synaptic terminals. Patch-clamp studies in brain slices and astrocyte/neuron co-cultures have provided functional evidence that uninterrupted Gln synthesis in astrocytes and its transport to neurons, as mediated by specific carriers, promotes glutamatergic and GABA-ergic transmission. Gln entry into the neuronal compartment is facilitated by its abundance in the extracellular spaces relative to other amino acids. Gln also appears to affect neurotransmission directly by interacting with the NMDA class of Glu receptors. Transmission may also be modulated by alterations in cell membrane polarity related to the electrogenic nature of Gln transport or to uncoupled ion conductances in the neuronal or glial cell membranes elicited by Gln transporters. In addition, Gln appears to modulate the synthesis of the gaseous messenger, nitric oxide (NO), by controlling the supply to the cells of its precursor, arginine. Disturbances of Gln metabolism and/or transport contribute to changes in Glu-ergic or GABA-ergic transmission associated with different pathological conditions of the brain, which are best recognized in epilepsy, hepatic encephalopathy and manganese encephalopathy.

Methylmercury induces acute oxidative stress, altering Nrf2 protein level in primary microglial cells

The neurotoxicity of methylmercury (MeHg) is well documented in both humans and animals. MeHg causes acute and chronic damage to multiple organs, most profoundly the central nervous system (CNS). Microglial cells are derived from macrophage cell lineage, making up approximately 12% of cells in the CNS, yet their role in MeHg-induced neurotoxicity is not well defined. The purpose of the present study was to characterize microglial vulnerability to MeHg and their potential adaptive response to acute MeHg exposure. We examined the effects of MeHg on microglial viability, reactive oxygen species (ROS) generation, glutathione (GSH) level, redox homeostasis, and Nrf2 protein expression. Our data showed that MeHg (1-5 microM) treatment caused a rapid (within 1 min) concentration- and time-dependent increase in ROS generation, accompanied by a statistically significant decrease in the ratio of GSH and its oxidized form glutathione disulfide (GSSG) (GSH:GSSG ratio). MeHg increased the cytosolic Nrf2 protein level within 1 min of exposure, followed by its nuclear translocation after 10 min of treatment. Consistent with the nuclear translocation of Nrf2, quantitative real-time PCR revealed a concentration-dependent increase in the messenger RNA level of Ho-1, Nqo1, and xCT 30 min post MeHg exposure, whereas Nrf2 knockdown greatly reduced the upregulation of these genes. Furthermore, we observed increased microglial death upon Nrf2 knockdown by the small hairpin RNA approach. Taken together, our study has demonstrated that microglial cells are exquisitely sensitive to MeHg and respond rapidly to MeHg by upregulating the Nrf2-mediated antioxidant response.

Comparative study on the response of rat primary astrocytes and microglia to methylmercury toxicity

As the two major glial cell types in the brain, astrocytes and microglia play pivotal but different roles in maintaining optimal brain function. Although both cell types have been implicated as major targets of methylmercury (MeHg), their sensitivities and adaptive responses to this metal can vary given their distinctive properties and physiological functions. This study was carried out to compare the responses of astrocytes and microglia following MeHg treatment, specifically addressing the effects of MeHg on cell viability, reactive oxygen species (ROS) generation and glutathione (GSH) levels, as well as mercury (Hg) uptake and the expression of NF‐E2‐related factor 2 (Nrf2). Results showed that microglia are more sensitive to MeHg than astrocytes, a finding that is consistent with their higher Hg uptake and lower basal GSH levels. Microglia also demonstrated higher ROS generation compared with astrocytes. Nrf2 and its downstream genes were upregulated in both cell types, but with different kinetics (much faster in microglia). In summary, microglia and astrocytes each exhibit a distinct sensitivity to MeHg, resulting in their differential temporal adaptive responses. These unique sensitivities appear to be dependent on the cellular thiol status of the particular cell type.

Role of astrocytes in manganese mediated neurotoxicity

Astrocytes are responsible for numerous aspects of metabolic support, nutrition, control of the ion and neurotransmitter environment in central nervous system (CNS). Failure by astrocytes to support essential neuronal metabolic requirements plays a fundamental role in the pathogenesis of brain injury and the ensuing neuronal death. Astrocyte-neuron interactions play a central role in brain homeostasis, in particular via neurotransmitter recycling functions. Disruption of the glutamine (Gln)/glutamate (Glu) -γ-aminobutyric acid (GABA) cycle (GGC) between astrocytes and neurons contributes to changes in Glu-ergic and/or GABA-ergic transmission, and is associated with several neuropathological conditions, including manganese (Mn) toxicity. In this review, we discuss recent advances in support of the important roles for astrocytes in normal as well as neuropathological conditions primarily those caused by exposure to Mn.

GPR30 Regulates Glutamate Transporter GLT-1 Expression in Rat Primary Astrocytes

Background: Estrogen increases glutamate transporter GLT-1 expression. Results: G1, a selective agonist of GPR30, significantly increased GLT-1 expression in rat primary astrocytes, and GPR30 suppression reduced GLT-1 expression. Conclusion: The G protein-coupled estrogen receptor GPR30 plays a critical role in the regulation of GLT-1 expression. Significance: Understanding the mechanism of GPR30 regulation on GLT-1 expression is important to develop therapeutics against excitatory neuronal injury. The G protein-coupled estrogen receptor GPR30 contributes to the neuroprotective effects of 17β-estradiol (E2); however, the mechanisms associated with this protection have yet to be elucidated. Given that E2 increases astrocytic expression of glutamate transporter-1 (GLT-1), which would prevent excitotoxic-induced neuronal death, we proposed that GPR30 mediates E2 action on GLT-1 expression. To investigate this hypothesis, we examined the effects of G1, a selective agonist of GPR30, and GPR30 siRNA on astrocytic GLT-1 expression, as well as glutamate uptake in rat primary astrocytes, and explored potential signaling pathways linking GPR30 to GLT-1. G1 increased GLT-1 protein and mRNA levels, subject to regulation by both MAPK and PI3K signaling. Inhibition of TGF-α receptor suppressed the G1-induced increase in GLT-1 expression. Silencing GPR30 reduced the expression of both GLT-1 and TGF-α and abrogated the G1-induced increase in GLT-1 expression. Moreover, the G1-induced increase in GLT-1 protein expression was abolished by a protein kinase A inhibitor and an NF-κB inhibitor. G1 also enhanced cAMP response element-binding protein (CREB), as well as both NF-κB p50 and NF-κB p65 binding to the GLT-1 promoter. Finally, to model dysfunction of glutamate transporters, manganese was used, and G1 was found to attenuate manganese-induced impairment in GLT-1 protein expression and glutamate uptake. Taken together, the present data demonstrate that activation of GPR30 increases GLT-1 expression via multiple pathways, suggesting that GPR30 is worthwhile as a potential target to be explored for developing therapeutics of excitotoxic neuronal injury.

Manganese disrupts astrocyte glutamine transporter expression and function

Glutamine (Gln) plays an important role in brain energy metabolism and as a precursor for the synthesis of neurotransmitter glutamate and GABA. Previous studies have shown that astrocytic Gln transport is impaired following manganese (Mn) exposure. The present studies were performed to identify the transport routes and the respective Gln transporters contributing to the impairment. Rat neonatal cortical primary astrocytes treated with Mn displayed a significant decrease in Gln uptake mediated by the principle Gln transporting systems, N and ASC. Moreover, systems N, ASC and L were less efficient in Gln export after Mn treatment. Mn treatment caused a significant reduction of both in mRNA expression and protein levels of SNAT3 (system N), SNAT2 (system A) and LAT2 (system L), and lowered the protein but not mRNA expression of ASCT2 (system ASC). Mn exposure did not affect the expression of the less abundant systems N transporter SNAT5 and the system L transporter LAT1, at either the mRNA or protein level. Hence, Mn‐induced decrease of inward and outward Gln transport can be largely ascribed to the loss of the specific Gln transporters. Consequently, deregulation of glutamate homeostasis and its diminished availability to neurons may lead to impairment in glutamatergic neurotransmission, a phenomenon characteristic of Mn‐induced neurotoxicity.

Transforming growth factor-α mediates estrogen-induced upregulation of glutamate transporter GLT-1 in rat primary astrocytes.

Glutamate transporter‐1 (GLT‐1) plays a central role in preventing excitotoxicity by removing excess glutamate from the synaptic clefts. 17β‐Estradiol (E2) and tamoxifen (TX), a selective estrogen receptor (ER) modulator, afford neuroprotection in a range of experimental models. However, the mechanisms that mediate E2 and TX neuroprotection have yet to be elucidated. We tested the hypothesis that E2 and TX enhance GLT‐1 function by increasing transforming growth factor (TGF)‐α expression and, thus, attenuate manganese (Mn)‐induced impairment in astrocytic GLT‐1 expression and glutamate uptake in rat neonatal primary astrocytes. The results showed that E2 (10 nM) and TX (1 μM) increased GLT‐1 expression and reversed the Mn‐induced reduction in GLT‐1, both at the mRNA and protein levels. E2/TX also concomitantly reversed the Mn‐induced inhibition of astrocytic glutamate uptake. E2/TX activated the GLT‐1 promoter and attenuated the Mn‐induced repression of the GLT‐1 promoter in astrocytes. TGF‐α knockdown (siRNA) abolished the E2/TX effect on GLT‐1 expression, and inhibition of epidermal growth factor receptor (TGF‐α receptor) suppressed the effect of E2/TX on GLT‐1 expression and GLT‐1 promoter activity. E2/TX also increased TGF‐α mRNA and protein levels with a concomitant increase in astrocytic glutamate uptake. All ERs (ER‐α, ER‐β, and G protein‐coupled receptor 30) were involved in mediating E2 effects on the regulation of TGF‐α, GLT‐1, and glutamate uptake. These results indicate that E2/TX increases GLT‐1 expression in astrocytes via TGF‐α signaling, thus offering an important putative target for the development of novel therapeutics for neurological disorders.

Disruption of Astrocytic Glutamine Turnover by Manganese Is Mediated by the Protein Kinase C Pathway

Manganese (Mn) is a trace element essential for normal human development and is required for the proper functioning of a variety of physiological processes. Chronic exposure to Mn can cause manganism, a neurodegenerative disorder resembling idiopathic Parkinsons disease (PD). Mn(II) neurotoxicity is characterized by astrocytic impairment both in the expression and activity of glutamine (Gln) transporters. Because protein kinase C (PKC) activation leads to the downregulation of a number of neurotransmitter transporters and Mn(II) increases PKC activity, we hypothesized that the PKC signaling pathway contributes to the Mn(II)‐mediated disruption of Gln turnover. Our results have shown that Mn exposure increases the phosphorylation of both the PKCα and PKCδ isoforms. PKC activity was also shown to be increased in response to Mn(II) treatment. Corroborating our earlier observations, Mn(II) also caused a decrease in Gln uptake. This effect was blocked by PKC inhibitors. Notably, PKC activation caused a decrease in Gln uptake mediated by systems ASC and N, but had no effect on the activities of systems A and L. Exposure to α‐phorbol 12‐myristate 13‐acetate significantly decreased SNAT3 (system N) and ASCT2 (system ASC) protein levels. Additionally, a co‐immunoprecipitation studydemonstrated the association of SNAT3 and ASCT2 with the PKCδ isoform, and Western blotting revealed the Mn(II)‐mediated activation of PKCδ by proteolytic cleavage. PKC activation was also found to increase SNAT3 and ubiquitin ligase Nedd4‐2 binding and to induce hyperubiquitination. Taken together, these findings demonstrate that the Mn(II)‐induced dysregulation of Gln homeostasis in astrocytes involves PKCδ signaling accompanied by an increase in ubiquitin‐mediated proteolysis.