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.     

Specific association of c‐Jun‐like immunoreactivity but not c‐Jun p39 with normal and induced programmed cell death in the chick embryo

We have examined c‐Jun protein expression by immunocytochemistry in normal and pathologically induced cell death by focusing primarily on the developing neuromuscular system of the chick embryo. Several commercially available antibodies against c‐Jun were used in combination with the TUNEL technique or propidium iodide staining for detection of cells undergoing programmed cell death (PCD). Among these, a rabbit polyclonal antibody raised against the amino acids 91‐105 mapping to the amino terminal domain of mouse c‐Jun p39 (c‐Jun/sc45) transiently immunostained the cytoplasm of dying spinal cord motoneurons at a time coincident with naturally occurring motoneuron death. Late apoptotic bodies were devoid of c‐Jun/sc45 immunoreactivity. A monoclonal antibody directed against a region corresponding to the amino acids 26‐175 of c‐Jun p39 (c‐Jun/mAB) did not specifically immunostain dying neurons, but, rather, showed nuclear immunolabeling in almost all healthy motoneurons. Experimentally induced programmed death of motoneurons by means of early limb bud ablation, axotomy, or in ovo injection of the neurotoxin β‐bungarotoxin increased the number of dying cells showing positive c‐Jun/sc45 immunoreactivity. Immunoelectron microscopy with c‐Jun/sc45 antibody showed that the signal was present in the cytoplasm without a specific association with organelles, and was also present in large lysosome‐like dense bodies inside neuritic profiles. Similar findings were obtained in different types of cells undergoing normal or experimentally induced PCD. These include dorsal root ganglion neurons, Schwann cells, muscle cells, neural tube and neural crest cells during the earliest stages of spinal cord development, and interdigital mesenchymal cells of hindlimbs. In all these cases, cells showed morphological and histochemical characteristics of apoptotic‐like PCD. By contrast, motoneurons undergoing necrotic cell death induced by the excitotoxin N‐methyl‐D ‐aspartate did not show detectable c‐Jun/sc45 immunoreactivity, although they displayed an increase in nuclear c‐Jun/mAB immunostaining. In Western blot analysis of spinal cord extracts, c‐Jun/sc45 antibody weakly detected a 39‐kD band, corresponding to c‐Jun, and more strongly detected two additional bands of 66 and 45 kD which followed developmental changes coincident with naturally occurring or experimentally stimulated apoptotic motoneuron death. By contrast, c‐Jun/mAB only recognized a single p39 band as expected for c‐Jun, and did not display changes associated with neuronal apoptosis. From these data, we conclude that the c‐Jun/sc45 antibody recognizes apoptosis‐related proteins associated with the early stages of morphological PCD in a variety of neuronal and nonneuronal cells, and that c‐Jun/sc45 is a reliable marker for a variety of developing cells undergoing programmed cell death. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 171–190, 1999     

The Tau/A152T mutation,a risk factor for frontotemporal‐spectrum disorders,leads to NR2B receptor‐mediated excitotoxicity

We report on a novel transgenic mouse model expressing human full‐length Tau with the Tau mutation A152T (hTau^AT), a risk factor for FTD‐spectrum disorders including PSP and CBD. Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis‐sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short‐ or long‐term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage‐gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau^AT causes excitotoxicity mediated by NR2B‐containing NMDA receptors due to enhanced extracellular glutamate.     

In vivo inhibition of the mitochondrial H+‐ATP synthase in neurons promotes metabolic preconditioning

A key transducer in energy conservation and signaling cell death is the mitochondrial H⁺‐ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H⁺‐ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H⁺‐ATP synthase in cell death in vivo. The expression of hIF1 inhibits the activity of oxidative phosphorylation and mediates the shift of neurons to an enhanced aerobic glycolysis. Metabolic reprogramming induces brain preconditioning affording protection against quinolinic acid‐induced excitotoxicity. Mechanistically, preconditioning involves the activation of the Akt/p70S6K and PARP repair pathways and Bcl‐xL protection from cell death. Overall, our findings provide the first in vivo evidence highlighting the H⁺‐ATP synthase as a target to prevent neuronal cell death.     

Microglia in juvenile neuronal ceroid lipofuscinosis are primed toward a pro‐inflammatory phenotype

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a lysosomal storage disease caused by an autosomal recessive mutation in CLN3. Regions of microglial activation precede and predict areas of neuronal loss in JNCL; however, the functional role of activated microglia remains to be defined. The inflammasome is a key molecular pathway for activating pro‐IL‐1β in microglia, and IL‐1β is elevated in the brains of JNCL patients and can induce neuronal cell death. Here, we utilized primary microglia isolated from CLN3^Δex7/8 mutant and wild‐type (WT) mice to examine the impact of CLN3 mutation on microglial activation and inflammasome function. Treatment with neuronal lysates and ceramide, a lipid intermediate elevated in the JNCL brain, led to inflammasome activation and IL‐1β release in CLN3^Δex7/8 microglia but not WT cells, as well as increased expression of additional pro‐inflammatory mediators. Similar effects were observed following either TNF‐α or IL‐1β treatment, suggesting that CLN3^Δex7/8 microglia exist in primed state and hyper‐respond to several inflammatory stimuli compared to WT cells. CLN3^Δex7/8 microglia displayed constitutive caspase‐1 activity that when blocked led to increased glutamate release that coincided with hemichannel opening. Conditioned medium from activated CLN3^Δex7/8 or WT microglia induced significant cell death in CLN3^Δex7/8 but not WT neurons, demonstrating that intrinsically diseased CLN3^Δex7/8 neurons are less equipped to withstand cytotoxic insults generated by activated microglia. Collectively, aberrant microglial activation may contribute to the pathological chain of events leading to neurodegeneration during later stages of JNCL.

     

Inhibiting pro-death NMDA receptor signaling dependent on the NR2 PDZ ligand may not affect synaptic function or synaptic NMDA receptor signaling to gene expression

NMDA receptors (NMDARs) mediate ischemic brain damage, in part through interactions of the PDZ ligand of NR2 subunits with the PDZ domain proteins PSD-95 and neuronal nitric oxide synthase located within the NMDAR signaling complex. We have recently shown that this PDZ ligand-dependent pathway promotes neuronal death via p38 activation. A peptide mimetic of the NR2B PDZ ligand (TAT-NR2B9c) reduces p38-mediated death in vitro and p38-dependent ischemic damage in vivo. In the absence of the PDZ ligand-p38 pathway, such as in TAT-NR2B9c-treated neurons, or in NMDAR-expressing non-neuronal cells, NMDAR-dependent excitotoxicity is mediated largely by JNK and requires greater Ca2+ influx. A major reason for blocking pro-death signaling events downstream of the NMDAR as an anti-excitotoxic strategy is that it may spare physiological synaptic function and signaling. We find that neuroprotective doses of TAT-NR2B9c do not alter the frequency of spontaneous synaptic events within networks of cultured cortical neurons nor is mini-EPSC frequency altered. Furthermore, TAT-NR2B9c does not inhibit the capacity of synaptic NMDAR activity to promote neuroprotective changes in gene expression, including the up-regulation of PACAP via CREB, and suppression of the pro-oxidative FOXO target gene Txnip. Thus, while the NR2 PDZ ligand does not account for all the excitotoxic effects of excessive NMDAR activity, these findings underline the value of the specific targeting of death pathways downstream of the NMDAR.     

Susceptibility to excitotoxicity in aged hippocampal cultures and neuroprotection by non‐steroidal anti‐inflammatory drugs: role of mitochondrial calcium

Brain damage after insult and cognitive decline are related to excitotoxicity and strongly influenced by aging, yet mechanisms of aging‐dependent susceptibility to excitotoxicity are poorly known. Several non‐steroidal anti‐inflammatory drugs (NSAIDs) may prevent excitotoxicity and cognitive decline in the elderly by an unknown mechanism. Interestingly, after several weeks in vitro, hippocampal neurons display important hallmarks of neuronal aging in vivo. Accordingly, rat hippocampal neurons cultured for several weeks were used to investigate mechanisms of aging‐related susceptibility to excitotoxicity and neuroprotection by NSAIDs. We found that NMDA increased cytosolic Ca²⁺ concentration in young, mature and aged neurons but only promoted apoptosis in aged neurons. Resting Ca²⁺ levels and responses to NMDA increased with time in culture which correlated with changes in expression of NMDA receptor subunits. In addition, NMDA promoted mitochondrial Ca²⁺ uptake only in aged cultures. Consistently, specific inhibition of mitochondrial Ca²⁺ uptake decreased apoptosis. Finally, we found that a series of NSAIDs depolarized mitochondria and inhibited mitochondrial Ca²⁺ overload, thus preventing NMDA‐induced apoptosis in aged cultures. We conclude that mitochondrial Ca²⁺ uptake is critical for age‐related susceptibility to excitotoxicity and neuroprotection by NSAIDs.

     

The role of action potentials in determining neuron‐type‐specific responses to nitric oxide

The electrical activity in developing and mature neurons determines the intracellular calcium concentration ([Ca²⁺]_i), which in turn is translated into biochemical activities through various signaling cascades. Electrical activity is under control of neuromodulators, which can alter neuronal responses to incoming signals and increase the fidelity of neuronal communication. Conversely, the effects of neuromodulators can depend on the ongoing electrical activity within target neurons; however, these activity‐dependent effects of neuromodulators are less well understood. Here, we present evidence that the neuronal firing frequency and intrinsic properties of the action potential (AP) waveform set the [Ca²⁺]_i in growth cones and determine how neurons respond to the neuromodulator nitric oxide (NO). We used two well‐characterized neurons from the freshwater snail Helisoma trivolvis that show different growth cone morphological responses to NO: B5 neurons elongate filopodia, while those of B19 neurons do not. Combining whole‐cell patch clamp recordings with simultaneous calcium imaging, we show that the duration of an AP contributes to neuron‐specific differences in [Ca²⁺]_i, with shorter APs in B19 neurons yielding lower growth cone [Ca²⁺]_i. Through the partial inhibition of voltage‐gated K⁺ channels, we increased the B19 AP duration resulting in a significant increase in [Ca²⁺]_i that was then sufficient to cause filopodial elongation following NO treatment. Our results demonstrate a neuron‐type specific correlation between AP shape, [Ca²⁺]_i, and growth cone motility, providing an explanation to how growth cone responses to guidance cues depend on intrinsic electrical properties and helping explain the diverse effects of NO across neuronal populations. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 435–451, 2015     

P2X7 receptor‐induced death of motor neurons by a peroxynitrite/FAS‐dependent pathway

The P2X7 receptor/channel responds to extracellular ATP and is associated with neuronal death and neuroinflammation in spinal cord injury and amyotrophic lateral sclerosis. Whether activation of P2X7 directly causes motor neuron death is unknown. We found that cultured motor neurons isolated from embryonic rat spinal cord express P2X7 and underwent caspase‐dependent apoptosis when exposed to exceptionally low concentrations of the P2X7 agonist 2′(3′)‐O‐(4‐Benzoylbenzoyl)‐ATP. The P2X7 inhibitors BBG, oATP, and KN‐62 prevented 2′(3′)‐O‐(4‐Benzoylbenzoyl)‐ATP‐induced motor neuron death. The endogenous P2X7 agonist ATP induced motor neuron death at low concentrations (1‐100 μM). High concentrations of ATP (1 mM) paradoxically became protective due to degradation in the culture media to produce adenosine and activate adenosine receptors. P2X7‐induced motor neuron death was dependent on neuronal nitric oxide synthase‐mediated production of peroxynitrite, p38 activation, and autocrine FAS signaling. Taken together, our results indicate that motor neurons are highly sensitive to P2X7 activation, which triggers apoptosis by activation of the well‐established peroxynitrite/FAS death pathway in motor neurons.     

MFN2 Couples Glutamate Excitotoxicity and Mitochondrial Dysfunction in Motor Neurons

Mitochondrial dysfunction plays a central role in glutamate-evoked neuronal excitotoxicity, and mitochondrial fission/fusion dynamics are essential for mitochondrial morphology and function. Here, we establish a novel mechanistic linker among glutamate excitotoxicity, mitochondrial dynamics, and mitochondrial dysfunction in spinal cord motor neurons. Ca²⁺-dependent activation of the cysteine protease calpain in response to glutamate results in the degradation of a key mitochondrial outer membrane fusion regulator, mitofusin 2 (MFN2), and leads to MFN2-mediated mitochondrial fragmentation preceding glutamate-induced neuronal death. MFN2 deficiency impairs mitochondrial function, induces motor neuronal death, and renders motor neurons vulnerable to glutamate excitotoxicity. Conversely, MFN2 overexpression blocks glutamate-induced mitochondrial fragmentation, mitochondrial dysfunction, and/or neuronal death in spinal cord motor neurons both in vitro and in mice. The inhibition of calpain activation also alleviates glutamate-induced excitotoxicity of mitochondria and neurons. Overall, these results suggest that glutamate excitotoxicity causes mitochondrial dysfunction by impairing mitochondrial dynamics via calpain-mediated MFN2 degradation in motor neurons and thus present a molecular mechanism coupling glutamate excitotoxicity and mitochondrial dysfunction.     

Partial rescue of neural apoptosis in the Lurcher mutant mouse through elimination of tissue plasminogen activator

Lurcher is a spontaneous mouse mutant characterized by premature and aberrant apoptosis in the cerebellum. The phenotype has been shown to be caused by a point mutation in the delta2 glutamate receptor subunit gene that results in a large constitutive inward current, which has proved that endogenous excitotoxicity can lead to apoptotic cell death. Additional studies have suggested a direct link between this endogenous excitotoxicity and the activation of intracellular cell death enzymes. We have previously shown that excitotoxic neuronal degeneration elicited through exogenous insults (e.g. excitotoxins, stroke) is promoted by an extracellular cascade involving the serine protease tissue plasminogen activator (tPA). However, whether it is through necrotic or apoptotic mechanisms that this excitotoxic cell death occurs has remained contested. We describe the attenuation of the Lurcher cell death progression in tPA-deficient mice. Elimination of tPA delayed the apoptotic death of Purkinje and granule neurons in Lurcher mice, and reduced the phosphorylation of Jun and the activation of caspase 8. These results indicate that not only does tPA-promoted excitotoxic cell death proceed through a receptor-mediated apoptotic pathway, but that neuronal cell death in the Lurcher mouse is facilitated by extracellular cascades in addition to the already described intracellular pathways. Finally, these findings suggest that therapeutic benefits may be achieved for a wide variety of insults to the CNS by regulating tPA activity to preserve neuronal viability.     

Alterations in STriatal‐Enriched protein tyrosine Phosphatase expression,activation, and downstream signaling in early and late stages of the YAC128 Huntington's disease mouse model

Striatal neurodegeneration and synaptic dysfunction in Huntington's disease are mediated by the mutant huntingtin (mHtt) protein. MHtt disrupts calcium homeostasis and facilitates excitotoxicity, in part by altering NMDA receptor (NMDAR) trafficking and function. Pre‐symptomatic (excitotoxin‐sensitive) transgenic mice expressing full‐length human mHtt with 128 polyglutamine repeats (YAC128 Huntington's disease mice) show increased calpain activity and extrasynaptic NMDAR (Ex‐NMDAR) localization and signaling. Furthermore, Ex‐NMDAR stimulation facilitates excitotoxicity in wild‐type cortical neurons via calpain‐mediated cleavage of STriatal‐Enriched protein tyrosine Phosphatase 61 (STEP61). The cleavage product, STEP33, cannot dephosphorylate p38 mitogen‐activated protein kinase (MAPK), thereby augmenting apoptotic signaling. Here, we show elevated extrasynaptic calpain‐mediated cleavage of STEP61 and p38 phosphorylation, as well as STEP61 inactivation and reduced extracellular signal‐regulated protein kinase 1/2 phosphorylation (ERK1/2) in the striatum of 6‐week‐old, excitotoxin‐sensitive YAC128 mice. Calpain inhibition reduced basal and NMDA‐induced STEP61 cleavage. However, basal p38 phosphorylation was normalized by a peptide disrupting NMDAR‐post‐synaptic density protein‐95 (PSD‐95) binding but not by calpain inhibition. In 1‐year‐old excitotoxin‐resistant YAC128 mice, STEP33 levels were not elevated, but STEP61 inactivation and p38 and ERK 1/2 phosphorylation levels were increased. These results show that in YAC128 striatal tissue, enhanced NMDAR–PSD‐95 interactions contributes to elevated p38 signaling in early, excitotoxin‐sensitive stages, and suggest that STEP61 inactivation enhances MAPK signaling at late, excitotoxin‐resistant stages.

     

Ephrin‐A3 reverse signaling regulates hippocampal neuronal damage and astrocytic glutamate transport after transient global ischemia

Increasing evidence indicates that the Eph receptors and their ephrin ligands are involved in the regulation of interactions between neurons and astrocytes. Moreover, astrocytic ephrin‐A3 reverse signaling mediated by EphA4 receptors is necessary for controlling the abundance of glial glutamate transporters. However, the role of ephrin‐A3 reverse signaling in astrocytic function and neuronal death under ischemic conditions remains unclear. In the present study, we found that the EphA4 receptor and its ephrin‐A3 ligand, which were distributed in neurons and astrocytes, respectively, in the hippocampus showed a coincident up‐regulation of protein expression in the early stage of ischemia. Application of clustered EphA4 decreased the expressions of astrocytic glutamate transporters together with astrocytic glutamate uptake capacity through activating ephrin‐A3 reverse signaling. In consequence, neuronal loss was aggravated in the CA1 region of the hippocampus accompanied by impaired hippocampus‐dependent spatial memory when clustered EphA4 treatment was administered prior to transient global ischemia. These findings indicate that EphA4‐mediated ephrin‐A3 reverse signaling is a crucial mechanism for astrocytes to control glial glutamate transporters and prevent glutamate excitotoxicity under pathological conditions.

     

The octadecaneuropeptide ODN prevents 6‐hydroxydopamine‐induced apoptosis of cerebellar granule neurons through a PKC‐MAPK‐dependent pathway

Oxidative stress, induced by various neurodegenerative diseases, initiates a cascade of events leading to apoptosis, and thus plays a critical role in neuronal injury. In this study, we have investigated the potential neuroprotective effect of the octadecaneuropeptide (ODN) on 6‐hydroxydopamine (6‐OHDA)‐induced oxidative stress and apoptosis in cerebellar granule neurons (CGN). ODN, which is produced by astrocytes, is an endogenous ligand for both central‐type benzodiazepine receptors (CBR) and a metabotropic receptor. Incubation of neurons with subnanomolar concentrations of ODN (10^?18 to 10^?12 M) inhibited 6‐OHDA‐evoked cell death in a concentration‐dependent manner. The effect of ODN on neuronal survival was abrogated by the metabotropic receptor antagonist, cyclo_1–8[DLeu⁵]OP, but not by a CBR antagonist. ODN stimulated polyphosphoinositide turnover and ERK phosphorylation in CGN. The protective effect of ODN against 6‐OHDA toxicity involved the phospholipase C/ERK MAPK transduction cascade. 6‐OHDA treatment induced an accumulation of reactive oxygen species, an increase of the expression of the pro‐apoptotic gene Bax, a drop of the mitochondrial membrane potential and a stimulation of caspase‐3 activity. Exposure of 6‐OHDA‐treated cells to ODN blocked all the deleterious effects of the toxin. Taken together, these data demonstrate for the first time that ODN is a neuroprotective agent that prevents 6‐OHDA‐induced oxidative stress and apoptotic cell death.