Janna V. Denisova

Neuronal Gap Junction Coupling Is Regulated by Glutamate and Plays Critical Role in Cell Death during Neuronal Injury

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI), and epilepsy. The coupling of neurons by gap junctions (electrical synapses) increases during neuronal injury. We report here that the ischemic increase in neuronal gap junction coupling is regulated by glutamate via group II metabotropic glutamate receptors (mGluRs). Specifically, using electrotonic coupling, Western blots, and siRNA in the mouse somatosensory cortex in vivo and in vitro, we demonstrate that activation of group II mGluRs increases background levels of neuronal gap junction coupling and expression of connexin 36 (Cx36) (neuronal gap junction protein), and inactivation of group II mGluRs prevents the ischemia-mediated increases in the coupling and Cx36 expression. We also show that the regulation is via cAMP/PKA (cAMP-dependent protein kinase)-dependent signaling and posttranscriptional control of Cx36 expression and that other glutamate receptors are not involved in these regulatory mechanisms. Furthermore, using the analysis of neuronal death, we show that inactivation of group II mGluRs or genetic elimination of Cx36 both dramatically reduce ischemia-mediated neuronal death in vitro and in vivo. Similar results are obtained using in vitro models of TBI and epilepsy. Our results indicate that neuronal gap junction coupling is a critical component of glutamate-dependent neuronal death. They also suggest that causal link among group II mGluR function, neuronal gap junction coupling, and neuronal death has a universal character and operates in different types of neuronal injuries.

Neuronal gap junctions are required for NMDA receptor-mediated excitotoxicity: implications in ischemic stroke.

N-methyl-D-aspartate receptors (NMDARs) play an important role in cell survival versus cell death decisions during neuronal development, ischemia, trauma, and epilepsy. Coupling of neurons by electrical synapses (gap junctions) is high or increases in neuronal networks during all these conditions. In the developing CNS, neuronal gap junctions are critical for two different types of NMDAR-dependent cell death. However, whether neuronal gap junctions play a role in NMDAR-dependent neuronal death in the mature CNS was not known. Using Fluoro-Jade B staining, we show that a single intraperitoneal administration of NMDA (100 mg/kg) to adult wild-type mice induces neurodegeneration in three forebrain regions, including rostral dentate gyrus. However, the NMDAR-mediated neuronal death is prevented by pharmacological blockade of neuronal gap junctions (with mefloquine, 30 mg/kg) and does not occur in mice lacking neuronal gap junction protein, connexin 36. Using Western blots, electrophysiology, calcium imaging, and gas chromatography-mass spectrometry in wild-type and connexin 36 knockout mice, we show that the reduced level of neuronal death in knockout animals is not caused by the reduced expression of NMDARs, activity of NMDARs, or permeability of the blood-brain barrier to NMDA. In wild-type animals, this neuronal death is not caused by upregulation of connexin 36 by NMDA. Finally, pharmacological and genetic inactivation of neuronal gap junctions in mice also dramatically reduces neuronal death caused by photothrombotic focal cerebral ischemia. The results indicate that neuronal gap junctions are required for NMDAR-dependent excitotoxicity and play a critical role in ischemic neuronal death.

Interplay of Chemical Neurotransmitters Regulates Developmental Increase in Electrical Synapses

Coupling of neurons by electrical synapses (gap junctions) transiently increases in the mammalian CNS during development. We report here that the developmental increase in neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein) are regulated by an interplay between the activity of group II metabotropic glutamate receptors (mGluRs) and GABAA receptors. Specifically, using dye coupling, electrotonic coupling, Western blots and small interfering RNA in the rat and mouse hypothalamus and cortex in vivo and in vitro, we demonstrate that activation of group II mGluRs augments, and inactivation prevents, the developmental increase in neuronal gap junction coupling and Cx36 expression. However, changes in GABAA receptor activity have the opposite effects. The regulation by group II mGluRs is via cAMP/PKA-dependent signaling, and regulation by GABAA receptors is via Ca2+/PKC-dependent signaling. Furthermore, the receptor-mediated upregulation of Cx36 requires a neuron-restrictive silencer element in the Cx36 gene promoter, and the downregulation involves the 3′-untranslated region of the Cx36 mRNA, as shown using reverse-transcription quantitative real-time PCR and luciferase reporter activity analysis. In addition, the methyl thiazolyl tetrazolium analysis indicates that mechanisms for the developmental increase in neuronal gap junction coupling directly control the death/survival mechanisms in developing neurons. Together, the results suggest a multitiered strategy for chemical synapses in developmental regulation of electrical synapses.

Non-cholinergic excitation in neurons after a chronic glutamate receptor blockade.

Previous experiments revealed a dramatic increase in excitatory acetylcholine transmission in hypothalamic cultures during a chronic decrease in glutamate activity. Data suggested that in the absence of glutamate excitation, acetylcholine becomes the major excitatory neurotransmitter. However, non-cholinergic excitatory activity was also detected in some neurons. Here, using calcium imaging in hypothalamic cultures chronically subjected to the glutamate receptor blockade, we demonstrate the contribution of metabotropic glutamate receptors, P2-purinoreceptors, histamine receptors, adrenoreceptors, and gap junctions, but not nitric oxide to this non-cholinergic excitation. We also show that the sensitivity of neurons to receptor agonists is increased following the blockade. Data suggest that multiple components contribute to the excitatory activity in hypothalamic neurons during a long-term decrease in glutamate activity.

Neuronal gap junctions play a role in the secondary neuronal death following controlled cortical impact.

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI) and epilepsy. Recent studies in mice showed a critical role for neuronal gap junctions in NMDA receptor-mediated excitotoxicity and ischemia-mediated neuronal death. Here, using controlled cortical impact (CCI) in adult mice, as a model of TBI, and Fluoro-Jade B staining for analysis of neuronal death, we set to determine whether neuronal gap junctions play a role in the CCI-mediated secondary neuronal death. We report that 24h post-CCI, substantial neuronal death is detected in a number of brain regions outside the injury core, including the striatum. The striatal neuronal death is reduced both in wild-type mice by systemic administration of mefloquine (a relatively selective blocker of neuronal gap junctions) and in knockout mice lacking connexin 36 (neuronal gap junction protein). It is also reduced by inactivation of group II metabotropic glutamate receptors (with LY341495) which, as reported previously, control the rapid increase in neuronal gap junction coupling following different types of neuronal injury. The results suggest that neuronal gap junctions play a critical role in the CCI-induced secondary neuronal death.

Regulation of Cholinergic Phenotype in Developing Neurons

Specification of neurotransmitter phenotype is critical for neural circuit development and is influenced by intrinsic and extrinsic factors. Recent findings in rat hypothalamus in vitro suggest the role of neurotransmitter glutamate in the regulation of cholinergic phenotype. Here we extended our previous studies on the mechanisms of glutamate-dependent regulation of cholinergic phenotypic properties in hypothalamic neurons. Using immunocytochemistry, electrophysiology, and calcium imaging, we demonstrate that hypothalamic expression of choline acetyltransferase (the cholinergic marker) and responsiveness of neurons to acetylcholine (ACh) receptor agonists increase during chronic administration of an N-methyl-D-aspartate receptor (NMDAR) blocker, MK-801, in developing rats in vivo and genetic and pharmacological inactivation of NMDARs in mouse and rat developing neuronal cultures. In hypothalamic cultures, an inactivation of NMDA receptors also induces ACh-dependent synaptic activity, as do inactivations of PKA, ERK/MAPK, CREB, and NF-kappaB, which are known to be regulated by NMDA receptors. Interestingly, the increase in cholinergic properties in developing neurons that is induced by NMDAR blockade is prevented by the blockade of ACh receptors, suggesting that function of ACh receptor is required for the cholinergic up-regulation. Using dual recording of monosynaptic excitatory postsynaptic currents, we further demonstrate that chronic inactivation of ionotropic glutamate receptors induces the cholinergic phenotype in a subset of glutamatergic neurons. The phenotypic switch is partial as ACh and glutamate are coreleased. The results suggest that developing neurons may not only coexpress multiple transmitter phenotypes, but can also change the phenotypes following changes in signaling in neuronal circuits.

Use of calcium imaging for analysis of neuronal gap junction coupling

We recently used Western blots for connexin 36 and neuronal dye coupling with neurobiotin to measure developmental decrease in neuronal gap junction coupling in cell cultures. To ask whether Ca2+ imaging also can be used to measure changes in the amount of neuronal gap junction coupling, we defined a Ca2+ coupling coefficient as the percentage of neurons with bicuculline-induced increases in intracellular Ca2+ that are suppressed by blocking gap junctions. We demonstrate in rat and mouse hypothalamic neuronal cultures that the Ca2+ coupling coefficient decreases during culture development, this decrease is prevented by manipulations that also prevent developmental decrease in neuronal gap junction coupling, and the coefficient is low in cultures lacking connexin 36. The results indicate that Ca2+ imaging is a useful tool to quantify the amount of neuronal gap junction coupling in cultures.

Death of Neurons following Injury Requires Conductive Neuronal Gap Junction Channels but Not a Specific Connexin

Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury). In cultures prepared from wild-type mice, over-expression and knockdown of Cx36-containing gap junctions augmented and prevented, respectively, neuronal death from NMDAR-mediated excitotoxicity and ischemia. In cultures obtained form from Cx36 knockout mice, re-expression of functional gap junction channels, containing either neuronal Cx36 or non-neuronal Cx43 or Cx31, resulted in increased neuronal death following insult. In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect. Finally, the absence of ethidium bromide uptake in non-transduced wild-type neurons two hours following NMDAR excitotoxicity or ischemia suggested the absence of active endogenous hemichannels in those neurons. Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons. A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed.

A potential role for neuronal connexin 36 in the pathogenesis of amyotrophic lateral sclerosis

Neuronal gap junctional protein connexin 36 (Cx36) contributes to neuronal death following a range of acute brain insults such as ischemia, traumatic brain injury and epilepsy. Whether Cx36 contributes to neuronal death and pathological outcomes in chronic neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), is not known. We show here that the expression of Cx36 is significantly decreased in lumbar segments of the spinal cord of both human ALS subjects and SOD1G93A mice as compared to healthy human and wild-type mouse controls, respectively. In purified neuronal cultures prepared from the spinal cord of wild-type mice, knockdown of Cx36 reduces neuronal death caused by overexpression of the mutant human SOD1-G93A protein. Taken together, these data suggest a possible contribution of Cx36 to ALS pathogenesis. A perspective for the use of blockers of Cx36 gap junction channels for ALS therapy is discussed.