Michelle L. Olsen

Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington's disease model mice

Huntingtons disease (HD) is characterized by striatal medium spiny neuron (MSN) dysfunction, but the underlying mechanisms remain unclear. We explored roles for astrocytes, in which mutant huntingtin is expressed in HD patients and mouse models. We found that symptom onset in R6/2 and Q175 HD mouse models was not associated with classical astrogliosis, but was associated with decreased Kir4.1 K+ channel functional expression, leading to elevated in vivo striatal extracellular K+, which increased MSN excitability in vitro. Viral delivery of Kir4.1 channels to striatal astrocytes restored Kir4.1 function, normalized extracellular K+, ameliorated aspects of MSN dysfunction, prolonged survival and attenuated some motor phenotypes in R6/2 mice. These findings indicate that components of altered MSN excitability in HD may be caused by heretofore unknown disturbances of astrocyte-mediated K+ homeostasis, revealing astrocytes and Kir4.1 channels as therapeutic targets.

Functional implications for Kir4.1 channels in glial biology: from K+ buffering to cell differentiation

Astrocytes and oligodendrocytes are characterized by a very negative resting potential and a high resting permeability for K+ ions. Early pharmacological and biophysical studies suggested that the resting potential is established by the activity of inwardly rectifying, Ba2+ sensitive, weakly rectifying Kir channels. Molecular cloning has identified 16 Kir channels genes of which several mRNA transcripts and protein products have been identified in glial cells. However, genetic deletion and siRNA knock‐down studies suggest that the resting conductance of astrocytes and oligodendrocytes is largely due to Kir4.1. Loss of Kir4.1 causes membrane depolarization, and a break‐down of K+ and glutamate homeostasis which results in seizures and wide‐spread white matter pathology. Kir channels have also been shown to act as critical regulators of cell division whereby Kir function is correlated with an exit from the cell cycle. Conversely, loss of functional Kir channels is associated with re‐entry of cells into the cell cycle and gliosis. A loss of functional Kir channels has been shown in a number of neurological diseases including temporal lobe epilepsy, amyotrophic lateral sclerosis, retinal degeneration and malignant gliomas. In the latter, expression of Kir4.1 is sufficient to arrest the aberrant growth of these glial derived tumor cells. Kir4.1 therefore represents a potential therapeutic target in a wide variety of neurological conditions.

Methyl-CpG-binding protein 2 (MECP2) mutation type is associated with disease severity in Rett syndrome

Background Rett syndrome (RTT), a neurodevelopmental disorder that primarily affects girls, is characterised by a period of apparently normal development until 6–18 months of age when motor and communication abilities regress. More than 95% of individuals with RTT have mutations in methyl-CpG-binding protein 2 (MECP2), whose protein product modulates gene transcription. Surprisingly, although the disorder is caused by mutations in a single gene, disease severity in affected individuals can be quite variable. To explore the source of this phenotypic variability, we propose that specific MECP2 mutations lead to different degrees of disease severity. Methods Using a database of 1052 participants assessed over 4940 unique visits, the largest cohort of both typical and atypical RTT patients studied to date, we examined the relationship between MECP2 mutation status and various phenotypic measures over time. Results In general agreement with previous studies, we found that particular mutations, such as p.Arg133Cys, p.Arg294X, p.Arg306Cys, 3° truncations and other point mutations, were relatively less severe in both typical and atypical RTT. In contrast, p.Arg106Trp, p.Arg168X, p.Arg255X, p.Arg270X, splice sites, deletions, insertions and deletions were significantly more severe. We also demonstrated that, for most mutation types, clinical severity increases with age. Furthermore, of the clinical features of RTT, ambulation, hand use and age at onset of stereotypies are strongly linked to overall disease severity. Conclusions We have confirmed that MECP2 mutation type is a strong predictor of disease severity. These data also indicate that clinical severity continues to become progressively worse regardless of initial severity. These findings will allow clinicians and families to anticipate and prepare better for the needs of individuals with RTT.

ClC3 Is a Critical Regulator of the Cell Cycle in Normal and Malignant Glial Cells

Although most brain cells are postmitotic, small populations of progenitor or stem cells can divide throughout life. These cells are believed to be the most likely source for primary brain malignancies including gliomas. Such tumors share many common features with nonmalignant glial cells but, because of their insidious growth, form cancers that are typically incurable. In studying the growth regulation of these tumors, we recently discovered that glioma cell division is preceded by a cytoplasmic condensation that we called premitotic condensation (PMC). PMC represents an obligatory step in cell replication and is linked to chromatin condensation. If perturbed, the time required to complete a division is significantly prolonged. We now show that PMC is a feature shared more commonly among normal and malignant cells and that the reduction of cell volume is accomplished by Cl− efflux through ClC3 Cl− channels. Patch-clamp electrophysiology demonstrated a significant upregulation of chloride currents at M phase of the cell cycle. Colocalization studies and coimmunoprecipitation experiments showed the channel on the plasma membrane and at the mitotic spindle. To demonstrate a mechanistic role for ClC3 in PMC, we knocked down ClC3 expression using short hairpin RNA constructs. This resulted in a significant reduction of chloride currents at M phase that was associated with a decrease in the rate of PMC and a similar impairment of DNA condensation. These data suggest that PMC is an integral part of cell division and is dependent on ClC3 channel function.

Functional Expression of Kir4.1 Channels in Spinal Cord Astrocytes

Spinal cord astrocytes (SCA) have a high permeability to K+ and hence have hyperpolarized resting membrane potentials. The underlying K+ channels are believed to participate in the uptake of neuronally released K+. These K+ channels have been studied extensively with regard to their biophysics and pharmacology, but their molecular identity in spinal cord is currently unknown. Using a combination of approaches, we demonstrate that channels composed of the Kir4.1 subunit are responsible for mediating the resting K+ conductance in SCA. Biophysical analysis demonstrates astrocytic Kir currents as weakly rectifying, potentiated by increasing [K+]o, and inhibited by micromolar concentrations of Ba2+. These currents were insensitive to tolbutemide, a selective blocker of Kir6.x channels, and to tertiapin, a blocker for Kir1.1 and Kir3.1/3.4 channels. PCR and Western blot analysis show prominent expression of Kir4.1 in SCA, and immunocytochemistry shows localization Kir4.1 channels to the plasma membrane. Kir4.1 protein levels show a developmental upregulation in vivo that parallels an increase in currents recorded over the same time period. Kir4.1 is highly expressed throughout most areas of the gray matter in spinal cord in vivo and recordings from spinal cord slices show prominent Kir currents. Electrophysiological recordings comparing SCA of wild‐type mice with those of homozygote Kir4.1 knockout mice confirm a complete and selective absence of Kir channels in the knockout mice, suggesting that Kir4.1 is the principle channel mediating the resting K+ conductance in SCA in vitro and in situ.

BK Channels Are Linked to Inositol 1,4,5-Triphosphate Receptors via Lipid Rafts A NOVEL MECHANISM FOR COUPLING [Ca2+]i TO ION CHANNEL ACTIVATION

Glioma cells prominently express a unique splice variant of a large conductance, calcium-activated potassium channel (BK channel). These channels transduce changes in intracellular calcium to changes of K+ conductance in the cells and have been implicated in growth control of normal and malignant cells. The Ca2+ increase that facilitates channel activation is thought to occur via activation of intracellular calcium release pathways or influx of calcium through Ca2+-permeable ion channels. We show here that BK channel activation involves the activation of inositol 1,4,5-triphosphate receptors (IP3R), which localize near BK channels in specialized membrane domains called lipid rafts. Disruption of lipid rafts with methyl-β-cyclodextrin disrupts the functional association of BK channel and calcium source resulting in a >50% reduction in K+ conductance mediated by BK channels. The reduction of BK current by lipid raft disruption was overcome by the global elevation of intracellular calcium through inclusion of 750 nm Ca2+ in the pipette solution, indicating that neither the calcium sensitivity of the channel nor their overall number was altered. Additionally, pretreatment of glioma cells with 2-aminoethoxydiphenyl borate to inhibit IP3Rs negated the effect of methyl-β-cyclodextrin, providing further support that IP3Rs are the calcium source for BK channels. Taken together, these data suggest a privileged association of BK channels in lipid raft domains and provide evidence for a novel coupling of these Ca2+-sensitive channels to their second messenger source.

Mislocalization of Kir channels in malignant glia

Inwardly rectifying potassium (Kir) channels are a prominent feature of mature, postmitotic astrocytes. These channels are believed to set the resting membrane potential near the potassium equilibrium potential (EK) and are implicated in potassium buffering. A number of previous studies suggest that Kir channel expression is indicative of cell differentiation. We therefore set out to examine Kir channel expression in malignant glia, which are incapable of differentiation. We used two established and widely used glioma cell lines, D54MG (a WHO grade 4 glioma) and STTG‐1 (a WHO grade 3 glioma), and compared them to immature and differentiated astrocytes. Both glioma cell lines were characterized by large outward K+ currents, depolarized resting membrane potentials (Vm) (−38.5 ± 4.2 mV, D54 and −28.1 ± 3.5 mV, STTG1), and relatively high input resistances (Rm) (260.6 ± 64.7 MΩ, D54 and 687.2 ± 160.3 MΩ, STTG1). These features were reminiscent of immature astrocytes, which also displayed large outward K+ currents, had a mean Vm of −51.1 ± 3.7 and a mean Rm value of 627.5 ± 164 MΩ. In contrast, mature astrocytes had a significantly more negative resting membrane potential (−75.2 ± 0.56 mV), and a mean Rm of 25.4 ± 7.4 MΩ. Barium (Ba2+) sensitive Kir currents were >20‐fold larger in mature astrocytes (4.06 ± 1.1 nS/pF) than in glioma cells (0.169 ± 0.033 nS/pF D54, 0.244 ± 0.04 nS/pF STTG1), which had current densities closer to those of dividing, immature astrocytes (0.474 ± 0.12 nS/pF). Surprisingly, Western blot analysis shows expression of several Kir channel subunits in glioma cells (Kir2.3, 3.1, and 4.1). However, while in astrocytes these channels localize diffusely throughout the cell, in glioma cells they are found almost exclusively in either the cell nucleus (Kir2.3 and 4.1) or ER/Golgi (3.1). These data suggest that mislocalization of Kir channel proteins to intracellular compartments is responsible for a lack of appreciable Kir currents in glioma cells.

Spinal cord injury causes a wide-spread, persistent loss of Kir4.1 and glutamate transporter 1: benefit of 17β-oestradiol treatment

During neuronal activity astrocytes function to remove extracellular increases in potassium, which are largely mediated by the inwardly-rectifying potassium channel Kir4.1, and to take up excess glutamate via glutamate transporter 1, a glial-specific glutamate transporter. Here we demonstrate that expression of both of these proteins is reduced by nearly 80% following a crush spinal cord injury in adult male rats, 7 days post-injury. This loss extended to spinal segments several millimetres rostral and caudal to the lesion epicentre, and persisted at 4 weeks post-injury. Importantly, we demonstrate that loss of these two proteins is not a direct result of astrocyte loss, as immunohistochemistry at 7 days and western blots at 4 weeks demonstrate a marked up-regulation in glial fibrillary acidic protein expression. Kir4.1 and glutamate transporter 1 expression were partially rescued by post-spinal cord injury administration of physiological levels of 17beta-oestradiol (0.08 mg/kg/day) in vivo. Utilizing an in vitro culture system we demonstrate that 17beta-oestradiol treatment (50 nM) is sufficient to increase glutamate transporter 1 protein expression in spinal cord astrocytes. This increase in glutamate transporter 1 protein expression was reversed and Kir4.1 expression reduced in the presence of an oestrogen receptor antagonist, Fulvestrant 182,780 suggesting a direct translational regulation of Kir4.1 and glutamate transporter 1 via genomic oestrogen receptors. Using whole-cell patch-clamp recordings in cultured spinal cord astrocytes, we show that changes in protein expression following oestrogen application led to functional changes in Kir4.1 mediated currents. These findings suggest that the neuroprotective benefits previously seen with 17beta-oestradiol after spinal cord injury may be in part due to increased Kir4.1 and glutamate transporter 1 expression in astrocytes leading to improved potassium and glutamate homeostasis.

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.

The role of glial-specific Kir4.1 in normal and pathological states of the CNS.

Kir4.1 is an inwardly rectifying K+ channel expressed exclusively in glial cells in the central nervous system. In glia, Kir4.1 is implicated in several functions including extracellular K+ homeostasis, maintenance of astrocyte resting membrane potential, cell volume regulation, and facilitation of glutamate uptake. Knockout of Kir4.1 in rodent models leads to severe neurological deficits, including ataxia, seizures, sensorineural deafness, and early postnatal death. Accumulating evidence indicates that Kir4.1 plays an integral role in the central nervous system, prompting many laboratories to study the potential role that Kir4.1 plays in human disease. In this article, we review the growing evidence implicating Kir4.1 in a wide array of neurological disease. Recent literature suggests Kir4.1 dysfunction facilitates neuronal hyperexcitability and may contribute to epilepsy. Genetic screens demonstrate that mutations of KCNJ10, the gene encoding Kir4.1, causes SeSAME/EAST syndrome, which is characterized by early onset seizures, compromised verbal and motor skills, profound cognitive deficits, and salt-wasting. KCNJ10 has also been linked to developmental disorders including autism. Cerebral trauma, ischemia, and inflammation are all associated with decreased astrocytic Kir4.1 current amplitude and astrocytic dysfunction. Additionally, neurodegenerative diseases such as Alzheimer disease and amyotrophic lateral sclerosis demonstrate loss of Kir4.1. This is particularly exciting in the context of Huntington disease, another neurodegenerative disorder in which restoration of Kir4.1 ameliorated motor deficits, decreased medium spiny neuron hyperexcitability, and extended survival in mouse models. Understanding the expression and regulation of Kir4.1 will be critical in determining if this channel can be exploited for therapeutic benefit.