Browsing by Subject "HIPPOCAMPAL-NEURONS"

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  • Ludwig, Anastasia; Rivera Baeza, Claudio; Uvarov, Pavel (2017)
    Background: Cation-chloride cotransporters (CCCs) are indispensable for maintaining chloride homeostasis in multiple cell types, but K-Cl cotransporter KCC2 is the only CCC member with an exclusively neuronal expression in mammals. KCC2 is critical for rendering fast hyperpolarizing responses of ionotropic.-aminobutyric acid and glycine receptors in adult neurons, for neuronal migration in the developing central nervous system, and for the formation and maintenance of small dendritic protrusions-dendritic spines. Deficit in KCC2 expression and/or activity is associated with epilepsy and neuropathic pain, and effective strategies are required to search for novel drugs augmenting KCC2 function. Results: We revised current methods to develop a noninvasive optical approach for assessing KCC2 transport activity using a previously characterized genetically encoded chloride sensor. Our protocol directly assesses dynamics of KCC2-mediated chloride efflux and allows measuring genuine KCC2 activity with good spatial and temporal resolution. As a proof of concept, we used this approach to compare transport activities of the two known KCC2 splice isoforms, KCC2a and KCC2b, in mouse neuronal Neuro-2a cells. Conclusions: Our noninvasive optical protocol proved to be efficient for assessment of furosemide-sensitive chloride fluxes. Transport activities of the N-terminal splice isoforms KCC2a and KCC2b obtained by the novel approach matched to those reported previously using standard methods for measuring chloride fluxes.
  • Coleman, Sarah Kate; Cai, Chunlin; Kalkkinen, Nisse; Korpi, Esa R.; Keinanen, Kari (2010)
  • Bhandage, Amol K.; Jin, Zhe; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R.; Birnir, Bryndis (2014)
  • Bishop, Hannah I.; Cobb, Melanie M.; Kirmiz, Michael; Parajuli, Laxmi K.; Mandikian, Danielle; Philp, Ashleigh M.; Melnik, Mikhail; Kuja-Panula, Juha; Rauvala, Heikki; Shigemoto, Ryuichi; Murray, Karl D.; Trimmer, James S. (2018)
    Voltage-gated K+ (Kv) channels play important roles in regulating neuronal excitability. Kv channels comprise four principal alpha subunits, and transmembrane and/or cytoplasmic auxiliary subunits that modify diverse aspects of channel function. AMIGO-1, which mediates homophilic cell adhesion underlying neurite outgrowth and fasciculation during development, has recently been shown to be an auxiliary subunit of adult brain Kv2.1-containing Kv channels. We show that AMIGO-1 is extensively colocalized with both Kv2.1 and its paralog Kv2.2 in brain neurons across diverse mammals, and that in adult brain, there is no apparent population of AMIGO-1 outside of that colocalized with these Kv2 alpha subunits. AMIGO-1 is coclustered with Kv2 alpha subunits at specific plasma membrane (PM) sites associated with hypolemmal subsurface cisternae at neuronal ER:PM junctions. This distinct PM clustering of AMIGO-1 is not observed in brain neurons of mice lacking Kv2 alpha subunit expression. Moreover, in heterologous cells, coexpression of either Kv2.1 or Kv2.2 is sufficient to drive clustering of the otherwise uniformly expressed AMIGO-1. Kv2 alpha subunit coexpression also increases biosynthetic intracellular trafficking and PM expression of AMIGO-1 in heterologous cells, and analyses of Kv2.1 and Kv2.2 knockout mice show selective loss of AMIGO-1 expression and localization in neurons lacking the respective Kv2 alpha subunit. Together, these data suggest that in mammalian brain neurons, AMIGO-1 is exclusively associated with Kv2 alpha subunits, and that Kv2 alpha subunits are obligatory in determining the correct pattern of AMIGO-1 expression, PM trafficking and clustering.
  • Mavrovic, Martina; Uvarov, Pavel; Delpire, Eric; Vutskits, Laszlo; Kaila, Kai; Puskarjov, Martin (2020)
    KCC2, encoded in humans by the SLC12A5 gene, is a multifunctional neuron-specific protein initially identified as the chloride (Cl-) extruder critical for hyperpolarizing GABA(A) receptor currents. Independently of its canonical function as a K-Cl cotransporter, KCC2 regulates the actin cytoskeleton via molecular interactions mediated through its large intracellular C-terminal domain (CTD). Contrary to the common assumption that embryonic neocortical projection neurons express KCC2 at non-significant levels, here we show that loss of KCC2 enhances apoptosis of late-born upper-layer cortical projection neurons in the embryonic brain. In utero electroporation of plasmids encoding truncated, transport-dead KCC2 constructs retaining the CTD was as efficient as of that encoding full-length KCC2 in preventing elimination of migrating projection neurons upon conditional deletion of KCC2. This was in contrast to the effect of a full-length KCC2 construct bearing a CTD missense mutation (KCC2(R952H)), which disrupts cytoskeletal interactions and has been found in patients with neurological and psychiatric disorders, notably seizures and epilepsy. Together, our findings indicate ion transport-independent, CTD-mediated regulation of developmental apoptosis by KCC2 in migrating cortical projection neurons.
  • Mahadevan, Vivek; Khademullah, C. Sahara; Dargaei, Zahra; Chevrier, Jonah; Uvarov, Pavel; Kwan, Julian; Bagshaw, Richard D.; Pawson, Tony; Emili, Andrew; De Koninck, Yves; Anggono, Victor; Airaksinen, Matti; Woodin, Melanie A. (2017)
    KCC2 is a neuron-specific K+-Cl- cotransporter essential for establishing the Cl- gradient required for hyperpolarizing inhibition in the central nervous system (CNS). KCC2 is highly localized to excitatory synapses where it regulates spine morphogenesis and AMPA receptor confinement. Aberrant KCC2 function contributes to human neurological disorders including epilepsy and neuropathic pain. Using functional proteomics, we identified the KCC2-interactome in the mouse brain to determine KCC2-protein interactions that regulate KCC2 function. Our analysis revealed that KCC2 interacts with diverse proteins, and its most predominant interactors play important roles in postsynaptic receptor recycling. The most abundant KCC2 interactor is a neuronal endocytic regulatory protein termed PACSIN1 (SYNDAPI N1). We verified the PACSIN1KCC2 interaction biochemically and demonstrated that shRNA knockdown of PACSIN1 in hippocampal neurons increases KCC2 expression and hyperpolarizes the reversal potential for Cl-. Overall, our global native-KCC2 interactome and subsequent characterization revealed PACSIN1 as a novel and potent negative regulator of KCC2.
  • Khirug, Stanislav; Soni, Shetal; Saez Garcia, Marta; Tessier, Marine; Zhou, Liang; Kulesskaya, Natalia; Rauvala, Heikki; Lindholm, Dan; Ludwig, Anastasia; Molinari, Florence; Rivera, Claudio (2021)
    A striking result from epidemiological studies show a correlation between low alcohol intake and lower incidence for ischemic stroke and severity of derived brain injury. Although reduced apoptosis and inflammation has been suggested to be involved, little is known about the mechanism mediating this effect in vivo. Increase in intracellular chloride concentration and derived depolarizing GABAAR-mediated transmission are common consequences following various brain injuries and are caused by the abnormal expression levels of the chloride cotransporters NKCC1 and KCC2. Downstream pro-apoptotic signaling through p75NTR may link GABAA depolarization with post-injury neuronal apoptosis. Here, we show that changes in GABAergic signaling, Cl− homeostasis, and expression of chloride cotransporters in the post-traumatic mouse brain can be significantly reduced by administration of 3% ethanol to the drinking water. Ethanol-induced upregulation of KCC2 has a positive impact on neuronal survival, preserving a large part of the cortical peri-infarct zone, as well as preventing the massive post-ischemic upregulation of the pro-apoptotic protein p75NTR. Importantly, intracortical multisite in vivo recordings showed that ethanol treatment could significantly ameliorate stroke-induced reduction in cortical activity. This surprising finding discloses a pathway triggered by low concentration of ethanol as a novel therapeutically relevant target.
  • Sato, Sebastian Sulis; Artoni, Pietro; Landi, Silvia; Cozzolino, Olga; Parra, Riccardo; Pracucci, Enrico; Trovato, Francesco; Szczurkowska, Joanna; Luin, Stefano; Arosio, Daniele; Beltram, Fabio; Cancedda, Laura; Kaila, Kai; Ratto, Gian Michele (2017)
    Intracellular chloride ([Cl-](i)) andpH(pH(i)) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl-](i) and pH(i), but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl-and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl-](i) and pH(i) at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl-](i) and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic two-photon excitation protocol to simultaneously determine the changes of pHi and [Cl-](i) in response to hypercapnia and seizure activity.
  • Roussa, Eleni; Speer, Jan Manuel; Chudotvorova, Ilona; Khakipoor, Shokoufeh; Smirnov, Sergei; Rivera Baeza, Claudio; Krieglstein, Kerstin (2016)
    Functional activation of the neuronal K+-Cl- co-transporter KCC2 (also known as SLC12A5) is a prerequisite for shifting GABAA responses from depolarizing to hyperpolarizing during development. Here, we introduce transforming growth factor beta 2 (TGF-beta 2) as a new regulator of KCC2 membrane trafficking and functional activation. TGF-beta 2 controls membrane trafficking, surface expression and activity of KCC2 in developing and mature mouse primary hippocampal neurons, as determined by immunoblotting, immunofluorescence, biotinylation of surface proteins and KCC2-mediated Cl- extrusion. We also identify the signaling pathway from TGF-beta 2 to cAMP-response-element-binding protein (CREB) and Ras-associated binding protein 11b (Rab11b) as the underlying mechanism for TGF-beta 2-mediated KCC2 trafficking and functional activation. TGF-beta 2 increases colocalization and interaction of KCC2 with Rab11b, as determined by 3D stimulated emission depletion (STED) microscopy and co-immunoprecipitation, respectively, induces CREB phosphorylation, and enhances Rab11b gene expression. Loss of function of either CREB1 or Rab11b suppressed TGF-beta 2-dependent KCC2 trafficking, surface expression and functionality. Thus, TGF-beta 2 is a new regulatory factor for KCC2 functional activation and membrane trafficking, and a putative indispensable molecular determinant for the developmental shift of GABAergic transmission.
  • Di Lieto, Antonio; Rantamaki, Tomi; Vesa, Liisa; Yanpallewar, Sudhirkumar; Antila, Hanna; Lindholm, Jesse; Rios, Maribel; Tessarollo, Lino; Castren, Eero (2012)