Spermidine Synthase Localization in Retinal Layers: Early Age Changes.

Polyamine (PA) spermidine (SPD) plays a crucial role in aging. Since SPD accumulates in glial cells, particularly in Müller retinal cells (MCs), the expression of the SPD-synthesizing enzyme spermidine synthase (SpdS) in Müller glia and age-dependent SpdS activity are not known. We used immunocytochemistry, Western blot (WB), and image analysis on rat retinae at postnatal days 3, 21, and 120. The anti-glutamine synthetase (GS) antibody was used to identify glial cells. In the neonatal retina (postnatal day 3 (P3)), SpdS was expressed in almost all progenitor cells in the neuroblast. However, by day 21 (P21), the SpdS label was pronouncedly expressed in multiple neurons, while GS labels were observed only in radial Müller glial cells. During early cell adulthood, at postnatal day 120 (P120), SpdS was observed solely in ganglion cells and a few other neurons. Western blot and semi-quantitative analyses of SpdS labeling showed a dramatic decrease in SpdS at P21 and P120 compared to P3. In conclusion, the redistribution of SpdS with aging indicates that SPD is first synthesized in all progenitor cells and then later in neurons, but not in glia. However, MCs take up and accumulate SPD, regardless of the age-associated decrease in SPD synthesis in neurons.

Putrescine Intensifies Glu/GABA Exchange Mechanism and Promotes Early Termination of Seizures.

Endogenous anticonvulsant mechanisms represent a reliable and currently underdeveloped strategy against recurrent seizures and may recall novel original therapeutics. Here, we investigated whether the intensification of the astroglial Glu-GABA exchange mechanism by application of the GABA precursor putrescine (PUT) may be effective against convulsive and non-convulsive seizures. We explored the potential of PUT to inhibit spontaneous spike-and-wave discharges (SWDs) in WAG/Rij rats, a genetic model of absence epilepsy. Significant shortening of SWDs in response to intraperitoneally applied PUT has been observed, which could be antagonized by blocking GAT-2/3-mediated astrocytic GABA release with the specific inhibitor SNAP-5114. Direct application of exogenous GABA also reduced SWD duration, suggesting that PUT-triggered astroglial GABA release through GAT-2/3 may be a critical step in limiting seizure duration. PUT application also dose-dependently shortened seizure-like events (SLEs) in the low-[Mg2+] in vitro model of temporal lobe epilepsy. SNAP-5114 reversed the antiepileptic effect of PUT in the in vitro model as well, further confirming that PUT reduces seizure duration by triggering glial GABA release. In accordance, we observed that PUT specifically reduces the frequency of excitatory synaptic potentials, suggesting that it specifically acts at excitatory synapses. We also identified that PUT specifically eliminated the tonic depolarization-induced desynchronization of SLEs. Since PUT is an important source of glial GABA and we previously showed significant GABA release, it is suggested that the astroglial Glu-GABA exchange mechanism plays a key role in limiting ictal discharges, potentially opening up novel pathways to control seizure propagation and generalization.

The involvement of polyamine uptake and synthesis pathways in the proliferation of neonatal astrocytes.

Polyamines (PAs), such as spermidine (SPD) and spermine (SPM), are essential to promote cell growth, survival, proliferation, and longevity. In the adult central nervous system (CNS), SPD and SPM are accumulated predominantly in healthy adult glial cells where PA synthesis is not present. To date, the accumulation and biosynthesis of PAs in developing astrocytes are not well understood. The purpose of the present study was to determine the contribution of uptake and/or synthesis of PAs using proliferation of neonatal astrocytes as an endpoint. We inhibited synthesis of PAs using α-difluoromethylornithine (DFMO; an inhibitor of the PA biosynthetic enzyme ornithine decarboxylase (ODC)) and inhibited uptake of PAs using trimer44NMe (PTI; a novel polyamine transport inhibitor). DFMO, but not PTI alone, blocked proliferation, suggesting that PA biosynthesis was present. Furthermore, exogenous administration of SPD rescued cell proliferation when PA synthesis was blocked by DFMO. When both synthesis and uptake of PAs were inhibited (DFMO + PTI), exogenous SPD no longer supported proliferation. These data indicate that neonatal astrocytes synthesize sufficient quantities of PAs de novo to support cell proliferation, but are also able to import exogenous PAs. This suggests that the PA uptake mechanism is present in both neonates as well as in adults and can support cell proliferation in neonatal astrocytes when ODC is blocked.

Retinal adaptation to dim light vision in spectacled caimans (Caiman crocodilus fuscus): Analysis of retinal ultrastructure.

It has been shown that mammalian retinal glial (Müller) cells act as living optical fibers that guide the light through the retinal tissue to the photoreceptor cells (Agte et al., 2011; Franze et al., 2007). However, for nonmammalian species it is unclear whether Müller cells also improve the transretinal light transmission. Furthermore, for nonmammalian species there is a lack of ultrastructural data of the retinal cells, which, in general, delivers fundamental information of the retinal function, i.e. the vision of the species. A detailed study of the cellular ultrastructure provides a basic approach of the research. Thus, the aim of the present study was to investigate the retina of the spectacled caimans at electron and light microscopical levels to describe the structural features. For electron microscopy, we used a superfast microwave fixation procedure in order to achieve more precise ultrastructural information than common fixation techniques. As result, our detailed ultrastructural study of all retinal parts shows structural features which strongly indicate that the caiman retina is adapted to dim light and night vision. Various structural characteristics of Müller cells suppose that the Müller cell may increase the light intensity along the path of light through the neuroretina and, thus, increase the sensitivity of the scotopic vision of spectacled caimans. Müller cells traverse the whole thickness of the neuroretina and thus may guide the light from the inner retinal surface to the photoreceptor cell perikarya and the Müller cell microvilli between the photoreceptor segments. Thick Müller cell trunks/processes traverse the layers which contain light-scattering structures, i.e., nerve fibers and synapses. Large Müller cell somata run through the inner nuclear layer and contain flattened, elongated Müller cell nuclei which are arranged along the light path and, thus, may reduce the loss of the light intensity along the retinal light path. The oblique arrangement of many Müller cell trunks/processes in the inner plexiform layer and the large Müller cell somata in the inner nuclear layer may suggest that light guidance through Müller cells increases the visual sensitivity. Furthermore, an adaptation of the caiman retina to low light levels is strongly supported by detailed ultrastructural data of other retinal parts, e.g. by (i) the presence of a guanine-based retinal tapetum, (ii) the rod dominance of the retina, (iii) the presence of photoreceptor cell nuclei, which penetrate the outer limiting membrane, (iv) the relatively low densities of photoreceptor and neuronal cells which is compensated by (v) the presence of rods with long and thick outer segments, that may increase the probability of photon absorption. According to a cell number analysis, the central and temporal areas of the dorsal tapetal retina, which supports downward prey detection in darker water, are the sites of the highest diurnal contrast/color vision, i.e. cone vision and of the highest retinal light sensitivity, i.e. rod vision.

Müller glial cells contribute to dim light vision in the spectacled caiman (Caiman crocodilus fuscus): Analysis of retinal light transmission.

In this study, we show the capability of Müller glial cells to transport light through the inverted retina of reptiles, specifically the retina of the spectacled caimans. Thus, confirming that Müller cells of lower vertebrates also improve retinal light transmission. Confocal imaging of freshly isolated retinal wholemounts, that preserved the refractive index landscape of the tissue, indicated that the retina of the spectacled caiman is adapted for vision under dim light conditions. For light transmission experiments, we used a setup with two axially aligned objectives imaging the retina from both sides to project the light onto the inner (vitreal) surface and to detect the transmitted light behind the retina at the receptor layer. Simultaneously, a confocal microscope obtained images of the Müller cells embedded within the vital tissue. Projections of light onto several representative Müller cell trunks within the inner plexiform layer, i.e. (i) trunks with a straight orientation, (ii) trunks which are formed by the inner processes and (iii) trunks which get split into inner processes, were associated with increases in the intensity of the transmitted light. Projections of light onto the periphery of the Müller cell endfeet resulted in a lower intensity of transmitted light. In this way, retinal glial (Müller) cells support dim light vision by improving the signal-to-noise ratio which increases the sensitivity to light. The field of illuminated photoreceptors mainly include rods reflecting the rod dominance of the of tissue. A subpopulation of Müller cells with downstreaming cone cells led to a high-intensity illumination of the cones, while the surrounding rods were illuminated by light of lower intensity. Therefore, Müller cells that lie in front of cones may adapt the intensity of the transmitted light to the different sensitivities of cones and rods, presumably allowing a simultaneous vision with both receptor types under dim light conditions.

Novel KCNJ10 Gene Variations Compromise Function of Inwardly Rectifying Potassium Channel 4.1.

TheKCNJ10gene encoding Kir4.1 contains numerous SNPs whose molecular effects remain unknown. We investigated the functional consequences of uncharacterized SNPs (Q212R, L166Q, and G83V) on homomeric (Kir4.1) and heteromeric (Kir4.1-Kir5.1) channel function. We compared these with previously characterized EAST/SeSAME mutants (G77R and A167V) in kidney-derived tsA201 cells and in glial cell-derived C6 glioma cells. The membrane potentials of tsA201 cells expressing G77R and G83V were significantly depolarized as compared with WTKir4.1, whereas cells expressing Q212R, L166Q, and A167V were less affected. Furthermore, macroscopic currents from cells expressing WTKir4.1 and Q212R channels did not differ, whereas currents from cells expressing L166Q, G83V, G77R, and A167V were reduced. Unexpectedly, L166Q current responses were rescued when co-expressed with Kir5.1. In addition, we observed notable differences in channel activity between C6 glioma cells and tsA201 cells expressing L166Q and A167V, suggesting that there are underlying differences between cell lines in terms of Kir4.1 protein synthesis, stability, or expression at the surface. Finally, we determined spermine (SPM) sensitivity of these uncharacterized SNPs and found that Q212R-containing channels displayed reduced block by 1 µmSPM. At 100 µmSPM, the block was equal to or greater than WT, suggesting that the greater driving force of SPM allowed achievement of steady state. In contrast, L166Q-Kir5.1 channels achieved a higher block than WT, suggesting a more stable interaction of SPM in the deep pore cavity. Overall, our data suggest that G83V, L166Q, and Q212R residues play a pivotal role in controlling Kir4.1 channel function.

New Insights on Astrocyte Ion Channels: Critical for Homeostasis and Neuron-Glia Signaling.

Initial biophysical studies on glial cells nearly 50 years ago identified these cells as being electrically silent. These first studies also demonstrated a large K(+) conductance, which led to the notion that glia may regulate extracellular K(+) levels homeostatically. This view has now gained critical support from the study of multiple disease models discussed herein. Dysfunction of a major astrocyte K(+) channel, Kir4.1, appears as an early pathological event underlying neuronal phenotypes in several neurodevelopmental and neurodegenerative diseases. An expanding list of other astrocyte ion channels, including the calcium-activated ion channel BEST-1, hemichannels, and two-pore domain K(+) channels, all contribute to astrocyte biology and CNS function and underpin new forms of crosstalk between neurons and glia. Once considered merely the glue that holds the brain together, it is now increasingly recognized that astrocytes contribute in several fundamental ways to neuronal function. Emerging new insights and future perspectives of this active research area are highlighted within. SIGNIFICANCE STATEMENT: The critical role of astrocyte potassium channels in CNS homeostasis has been reemphasized by recent studies conducted in animal disease models. Emerging evidence also supports the signaling role mediated by astrocyte ion channels such as BEST1, hemichannels, and two-pore channels, which enable astrocytes to interact with neurons and regulate synaptic transmission and plasticity. This minisymposium highlights recent developments and future perspectives of these research areas.

Polyamine transport by the polyspecific organic cation transporters OCT1, OCT2, and OCT3.

Polyamines are ubiquitous organic cations implicated in many physiological processes. Because they are positively charged at physiological pH, carrier-mediated systems are necessary for effective membrane permeation, but the identity of specific polyamine transporter proteins in eukaryotic cells remains unclear. Polyspecific organic cation transporters (OCTs) interact with many natural and xenobiotic monovalent cations and have been reported to transport dicationic compounds, including the short polyamine putrescine. In this study, we used Xenopus oocytes expressing mammalian OCT1 (SLC22A1), OCT2 (SLC22A2), or OCT3 (SLC22A3) to assess binding and transport of longer-chain polyvalent polyamines. In OCT-expressing oocytes, [(3)H]MPP(+) uptake rates were 15- to 35-fold higher than in noninjected oocytes, whereas those for [(3)H]spermidine increased more modestly above the background, up to 3-fold. This reflected up to 20-fold lower affinity for spermidine than for MPP(+); thus, K(0.5) for MPP(+) was ~50 µM in OCT1, ~170 µM in OCT2, and ~60 µM in OCT3, whereas for spermidine, K(0.5) was ~1 mM in OCT1, OCT2, and OCT3. J(max) values for MPP(+) and spermidine were within the same range, suggesting that both compounds are transported at a similar turnover rate. To gain further insight into OCT substrate specificity, we screened a selection of structural polyamine analogues for effect on [(3)H]MPP(+) uptake. In general, blocking potency increased with overall hydrophobic character, which indicates that, as for monovalent cations, hydrophobicity is a major requirement for recognition in polyvalent OCT substrates and inhibitors. Our results demonstrate that the natural polyamines are low affinity, but relatively high turnover, substrates for OCTs. The identification of OCTs as polyamine transport systems may contribute to further understanding of the mechanisms involved in polyamine homeostasis and aid in the design of polyamine-like OCT-targeted drugs.

Changes in the Localization of Polyamine Spermidine in the Rat Retina with Age.

Polyamines (PAs) in the nervous system has a key role in regeneration and aging. Therefore, we investigated age-related changes in the expression of PA spermidine (SPD) in the rat retina. Fluorescent immunocytochemistry was used to evaluate the accumulation of SPD in retinae from rats of postnatal days 3, 21, and 120. Glial cells were identified using glutamine synthetase (GS), whereas DAPI, a marker of cell nuclei, was used to differentiate between retinal layers. SPD localization in the retina was strikingly different between neonates and adults. In the neonatal retina (postnatal day 3-P3), SPD is strongly expressed in practically all cell types, including radial glia and neurons. SPD staining showed strong co-localization with the glial marker GS in Müller Cells (MCs) in the outer neuroblast layer. In the weaning period (postnatal day 21-P21), the SPD label was strongly expressed in all MCs, but not in neurons. In early adulthood (postnatal day 120-P120), SPD was localized in MCs only and was co-localized with the glial marker GS. A decline in the expression of PAs in neurons was observed with age while glial cells accumulated SPD after the differentiation stage (P21) and during aging in MC cellular endfoot compartments.

As Verified with the Aid of Biotinylated Spermine, the Brain Cannot Take up Polyamines from the Bloodstream Leaving It Solely Dependent on Local Biosynthesis.

The importance of polyamines (PAs) for the central nervous system (CNS) is well known. Less clear, however, is where PAs in the brain are derived from. Principally, there are three possibilities: (i) intake by nutrition, release into the bloodstream, and subsequent uptake from CNS capillaries, (ii) production by parenchymatous organs, such as the liver, and again uptake from CNS capillaries, and (iii) uptake of precursors, such as arginine, from the blood and subsequent local biosynthesis of PAs within the CNS. The present investigation aimed to unequivocally answer the question of whether PAs, especially the higher ones like spermidine (SPD) and spermine (SPM), can or cannot be taken up into the brain from the bloodstream. For this purpose, a biotin-labelled analogue of spermine (B-X-SPM) was synthesized, characterized, and used to visualize its uptake into brain cells following application to acute brain slices, to the intraventricular space, or to the bloodstream. In acute brain slices there is strong uptake of B-X-SPM into protoplasmic and none in fibrous-type astrocytes. It is also taken up by neurons but to a lesser degree. Under in vivo conditions, astrocyte uptake of B-X-SPM from the brain interstitial fluid is also intense after intraventricular application. In contrast, following intracardial injection, there is no uptake from the bloodstream, indicating that the brain is completely dependent on the local synthesis of polyamines.

Unique Chemistry, Intake, and Metabolism of Polyamines in the Central Nervous System (CNS) and Its Body.

Polyamines (PAs) are small, versatile molecules with two or more nitrogen-containing positively charged groups and provide widespread biological functions. Most of these aspects are well known and covered by quite a number of excellent surveys. Here, the present review includes novel aspects and questions: (1) It summarizes the role of most natural and some important synthetic PAs. (2) It depicts PA uptake from nutrition and bacterial production in the intestinal system following loss of PAs via defecation. (3) It highlights the discrepancy between the high concentrations of PAs in the gut lumen and their low concentration in the blood plasma and cerebrospinal fluid, while concentrations in cellular cytoplasm are much higher. (4) The present review provides a novel and complete scheme for the biosynthesis of Pas, including glycine, glutamate, proline and others as PA precursors, and provides a hypothesis that the agmatine pathway may rescue putrescine production when ODC knockout seems to be lethal (solving the apparent contradiction in the literature). (5) It summarizes novel data on PA transport in brain glial cells explaining why these cells but not neurons preferentially accumulate PAs. (6) Finally, it provides a novel and complete scheme for PA interconversion, including hypusine, putreanine, and GABA (unique gliotransmitter) as end-products. Altogether, this review can serve as an updated contribution to understanding the PA mystery.

The Polyamine Spermine Potentiates the Propagation of Negatively Charged Molecules through the Astrocytic Syncytium.

The interest in astrocytes, the silent brain cells that accumulate polyamines (PAs), is growing. PAs exert anti-inflammatory, antioxidant, antidepressant, neuroprotective, and other beneficial effects, including increasing longevity in vivo. Unlike neurons, astrocytes are extensively coupled to others via connexin (Cx) gap junctions (GJs). Although there are striking modulatory effects of PAs on neuronal receptors and channels, PA regulation of the astrocytic GJs is not well understood. We studied GJ-propagation using molecules of different (i) electrical charge, (ii) structure, and (iii) molecular weight. Loading single astrocytes with patch pipettes containing membrane-impermeable dyes, we observed that (i) even small molecules do not easily permeate astrocytic GJs, (ii) the ratio of the charge to weight of these molecules is the key determinant of GJ permeation, (iii) the PA spermine (SPM) induced the propagation of negatively charged molecules via GJs, (iv) while no effects were observed on propagation of macromolecules with net-zero charge. The GJ uncoupler carbenoxolone (CBX) blocked such propagation. Taken together, these findings indicate that SPM is essential for astrocytic GJ communication and selectively facilitates intracellular propagation via GJs for negatively charged molecules through glial syncytium.

Molecular mechanisms of EAST/SeSAME syndrome mutations in Kir4.1 (KCNJ10).

Inwardly rectifying potassium channel Kir4.1 is critical for glial function, control of neuronal excitability, and systemic K(+) homeostasis. Novel mutations in Kir4.1 have been associated with EAST/SeSAME syndrome, characterized by mental retardation, ataxia, seizures, hearing loss, and renal salt waste. Patients are homozygous for R65P, G77R, C140R or T164I; or compound heterozygous for A167V/R297C or R65P/R199Stop, a deletion of the C-terminal half of the protein. We investigated the functional significance of these mutations by radiotracer efflux and inside-out membrane patch clamping in COSm6 cells expressing homomeric Kir4.1 or heteromeric Kir4.1/Kir5.1 channels. All of the mutations compromised channel function, but the underlying mechanisms were different. R65P, T164I, and R297C caused an alkaline shift in pH sensitivity, indicating that these positions are crucial for pH sensing and pore gating. In R297C, this was due to disruption of intersubunit salt bridge Glu(288)-Arg(297). C140R breaks the Cys(108)-Cys(140) disulfide bond essential for protein folding and function. A167V did not affect channel properties but may contribute to decreased surface expression in A167V/R297C. In G77R, introduction of a positive charge within the bilayer may affect channel structure or gating. R199Stop led to a dramatic decrease in surface expression, but channel activity was restored by co-expression with intact subunits, suggesting remarkable tolerance for truncation of the cytoplasmic domain. These results provide an explanation for the molecular defects that underlie the EAST/SeSAME syndrome.

Expression of the voltage- and Ca2+-dependent BK potassium channel subunits BKß1 and BKß4 in rodent astrocytes.

Large-conductance Ca(2+) -activated (BK) potassium channels are centrally involved in neurovascular coupling, immunity, and neural transmission. The ability to be synergistically activated by membrane depolarization, different ligands and intracellular Ca(2+) links intracellular signaling and membrane excitability. The diverse physiological functions of BK channels crucially depend on regulatory ß subunits. Although first studies characterized the neuronal distribution of BKß subunits in the rodent brain, it is largely unknown which ß subunit proteins are expressed in astrocytes and thus mediate these regulatory effects. We therefore analyzed the expression of BKß subunits in rat and mouse brain and glial cell cultures. A monospecific polyclonal antibody against the BKß4 channel subunit was raised, affinity-purified and extensively characterized. BKß4 and to a lesser degree BKß1 transcripts and protein were detected in several astrocytic populations and cultured cells. Particularly strong BKß4 immunostaining was detected in astrocytic progenitors derived from the subventricular zone. The overlapping expression of BKα and BKß4 in astrocytes implies a functional relationship and suggests that BKß4 is an important accessory ß subunit for astrocytic BK channels. In addition, BKß4 might exert effects independent of the α subunit as functional heterologous co-expression of Nav1.6 and BKß4 resulted in reduced Nav1.6 sodium currents. Thus, BKß4 expression in astrocytes likely participates in regulating astrocytic voltage gradients and maintaining K(+) homeostasis, hence enabling astrocytes to fulfill their complex regulatory influence on proper brain function.

Critical Role of Astrocytic Polyamine and GABA Metabolism in Epileptogenesis.

Accumulating evidence indicate that astrocytes are essential players of the excitatory and inhibitory signaling during normal and epileptiform activity via uptake and release of gliotransmitters, ions, and other substances. Polyamines can be regarded as gliotransmitters since they are almost exclusively stored in astrocytes and can be released by various mechanisms. The polyamine putrescine (PUT) is utilized to synthesize GABA, which can also be released from astrocytes and provide tonic inhibition on neurons. The polyamine spermine (SPM), synthesized form PUT through spermidine (SPD), is known to unblock astrocytic Cx43 gap junction channels and therefore facilitate astrocytic synchronization. In addition, SPM released from astrocytes may also modulate neuronal NMDA, AMPA, and kainate receptors. As a consequence, astrocytic polyamines possess the capability to significantly modulate epileptiform activity. In this study, we investigated different steps in polyamine metabolism and coupled GABA release to assess their potential to control seizure generation and maintenance in two different epilepsy models: the low-[Mg2+] model of temporal lobe epilepsy in vitro and in the WAG/Rij rat model of absence epilepsy in vivo. We show that SPM is a gliotransmitter that is released from astrocytes and significantly contributes to network excitation. Importantly, we found that inhibition of SPD synthesis completely prevented seizure generation in WAG/Rij rats. We hypothesize that this antiepileptic effect is attributed to the subsequent enhancement of PUT to GABA conversion in astrocytes, leading to GABA release through GAT-2/3 transporters. This interpretation is supported by the observation that antiepileptic potential of the Food and Drug Administration (FDA)-approved drug levetiracetam can be diminished by specifically blocking astrocytic GAT-2/3 with SNAP-5114, suggesting that levetiracetam exerts its effect by increasing surface expression of GAT-2/3. Our findings conclusively suggest that the major pathway through which astrocytic polyamines contribute to epileptiform activity is the production of GABA. Modulation of astrocytic polyamine levels, therefore, may serve for a more effective antiepileptic drug development in the future.

Uptake of Biotinylated Spermine in Astrocytes: Effect of Cx43 siRNA, HIV-Tat Protein and Polyamine Transport Inhibitor on Polyamine Uptake.

Polyamines (PAs) are polycationic biomolecules containing multiple amino groups. Patients with HIV-associated neurocognitive disorder (HAND) have high concentrations of the polyamine N-acetylated spermine in their brain and cerebral spinal fluid (CSF) and have increased PA release from astrocytes. These effects are due to the exposure to HIV-Tat. In healthy adult brain, PAs are accumulated but not synthesized in astrocytes, suggesting that PAs must enter astrocytes to be N-acetylated and released. Therefore, we tested if Cx43 hemichannels (Cx43-HCs) are pathways for PA flux in control and HIV-Tat-treated astrocytes. We used biotinylated spermine (b-SPM) to examine polyamine uptake. We found that control astrocytes and those treated with siRNA-Cx43 took up b-SPM, similarly suggesting that PA uptake is via a transporter/channel other than Cx43-HCs. Surprisingly, astrocytes pretreated with both HIV-Tat and siRNA-Cx43 showed increased accumulation of b-SPM. Using a novel polyamine transport inhibitor (PTI), trimer 44NMe, we blocked b-SPM uptake, showing that PA uptake is via a PTI-sensitive transport mechanism such as organic cation transporter. Our data suggest that Cx43 HCs are not a major pathway for b-SPM uptake in the condition of normal extracellular calcium concentration but may be involved in the release of PAs to the extracellular space during viral infection.

Activation of Glutamate Transporter-1 (GLT-1) Confers Sex-Dependent Neuroprotection in Brain Ischemia.

Stroke is one of the leading causes of long-term disability. During ischemic stroke, glutamate is released, reuptake processes are impaired, and glutamate promotes excitotoxic neuronal death. Astrocytic glutamate transporter 1 (GLT-1) is the major transporter responsible for removing excess glutamate from the extracellular space. A translational activator of GLT-1, LDN/OSU 0212320 (LDN) has been previously developed with beneficial outcomes in epileptic animal models but has never been tested as a potential therapeutic for ischemic strokes. The present study evaluated the effects of LDN on stroke-associated brain injury. Male and female mice received LDN or vehicle 24 h before or 2 h after focal ischemia was induced in the sensorimotor cortex. Sensorimotor performance was determined using the Rung Ladder Walk and infarct area was assessed using triphenyltetrazolium chloride staining. Males treated with LDN exhibited upregulated GLT-1 protein levels, significantly smaller infarct size, and displayed better sensorimotor performance in comparison to those treated with vehicle only. In contrast, there was no upregulation of GLT-1 protein levels and no difference in infarct size or sensorimotor performance between vehicle- and LDN-treated females. Taken together, our results indicate that the GLT-1 translational activator LDN improved stroke outcomes in young adult male, but not female mice.

Erratum: Tejeda-Bayron et al. Activation of Glutamate Transporter-1 (GLT-1) Confers Sex-Dependent Neuroprotection in Brain Ischemia. Brain Sci. 2021, 11, 76.

The authors wish to make the following corrections to this paper [...].

Potassium channel activity and glutamate uptake are impaired in astrocytes of seizure-susceptible DBA/2 mice.

PURPOSE: KCNJ10 encodes subunits of inward rectifying potassium (Kir) channel Kir4.1 found predominantly in glial cells within the brain. Genetic inactivation of these channels in glia impairs extracellular K(+) and glutamate clearance and produces a seizure phenotype. In both mice and humans, polymorphisms and mutations in the KCNJ10 gene have been associated with seizure susceptibility. The purpose of the present study was to determine whether there are differences in Kir channel activity and potassium- and glutamate-buffering capabilities between astrocytes from seizure resistant C57BL/6 (B6) and seizure susceptible DBA/2 (D2) mice that are consistent with an altered K(+) channel activity as a result of genetic polymorphism of KCNJ10. METHODS: Using cultured astrocytes and hippocampal brain slices together with whole-cell patch-clamp, we determined the electrophysiologic properties, particularly K(+) conductances, of B6 and D2 mouse astrocytes. Using a colorimetric assay, we determined glutamate clearance capacity by B6 and D2 astrocytes. RESULTS: Barium-sensitive Kir currents elicited from B6 astrocytes are substantially larger than those elicited from D2 astrocytes. In addition, potassium and glutamate buffering by D2 cortical astrocytes is impaired, relative to buffering by B6 astrocytes. DISCUSSION: In summary, the activity of Kir4.1 channels differs between seizure-susceptible D2 and seizure-resistant B6 mice. Reduced activity of Kir4.1 channels in astrocytes of D2 mice is associated with deficits in potassium and glutamate buffering. These deficits may, in part, explain the relatively low seizure threshold of D2 mice.

Kir4.1 potassium channel regulation via microRNA-205 in astrocytes exposed to hyperglycemic conditions.

Protecting neurons from neurotoxicity is a job mainly performed by astrocytes through glutamate uptake and potassium buffering. These functions are aided principally by the Kir4.1 inwardly rectifying potassium channels located in the membrane of astrocytes. Astrocytes grown in hyperglycemic conditions have decreased levels of Kir4.1 potassium channels as well as impaired potassium and glutamate uptake. Previous studies performed in a human corneal epithelial cell injury model demonstrated a mechanism of regulation of Kir4.1 expression via the binding of microRNA-250 (miR-205) to the Kir4.1 3´ untranslated region. Our purpose is to test if astrocytes express miR-205 and elucidate its role in regulating Kir4.1 expression in astrocytes grown in hyperglycemic conditions. We used quantitative-PCR to assess the levels of miR-205 in astrocytes grown in high glucose (25 mM) medium compared to astrocytes grown in normal glucose (5 mM). We found that not only was miR-205 expressed in astrocytes grown in normal glucose, but its expression was increased up to six-fold in astrocytes grown in hyperglycemic conditions. Transfection of miR-205 mimic or inhibitor was performed to alter the levels of miR-205 in astrocytes followed by western blot to assess Kir4.1 channel levels in these cells. Astrocytes treated with miR-205 mimic had a 38.6% reduction of Kir4.1 protein levels compared to control (mock-transfected) cells. In contrast, astrocytes transfected with miR-205 inhibitor were significantly upregulated compared to mock by 47.4%. Taken together, our data indicate that miR-205 negatively regulates the expression of Kir4.1 in astrocytes grown in hyperglycemic conditions.