Elevation In Neurons Research Articles

Neuronal ApoE4 stimulates C/EBPβ activation, promoting Alzheimer’s disease pathology in a mouse model

ApoE4 is a major genetic risk determinant for Alzheimer's disease (AD) and drives its pathogenesis via Aβ-dependent and -independent pathways. C/EBPβ, a proinflammatory cytokine-activated transcription factor, is upregulated in AD patients and increases cytokines and δ-secretase expression. Under physiological conditions, ApoE is mainly expressed in glial cells, but its neuronal expression is highly elevated under pathological stresses. However, how neuronal ApoE4 mediates AD pathologies remains incompletely understood. Here we show that ApoE4 activates C/EBPβ that subsequently regulates APP, Tau and BACE1 mRNA expression in mouse neurons, driving AD-like pathogenesis. To interrogate the pathological roles of both human ApoE4 and C/EBPβ elevation in neurons in the aged brain, we develop neuronal specific Thy1-ApoE4/C/EBPβ double transgenic mice. Neuronal ApoE4 strongly activates C/EBPβ and augmented δ-secretase subsequently cleaves increased mouse APP and Tau, promoting AD-like pathologies. Notably, Thy1-ApoE4/C/EBPβ mice develop amyloid deposits, Tau aggregates and neurodegeneration in an age-dependent manner, leading to synaptic dysfunction and cognitive disorders. Thus, our findings demonstrate that neuronal ApoE4 triggers AD pathogenesis via activating the crucial regulator C/EBPβ.

G-protein-coupled estrogen receptor activation upregulates interleukin-1 receptor antagonist in the hippocampus after global cerebral ischemia: implications for neuronal self-defense

BackgroundG-protein-coupled estrogen receptor (GPER/GPR30) is a novel membrane-associated estrogen receptor that can induce rapid kinase signaling in various cells. Activation of GPER can prevent hippocampal neuronal cell death following transient global cerebral ischemia (GCI), although the mechanisms remain unclear. In the current study, we sought to address whether GPER activation exerts potent anti-inflammatory effects in the rat hippocampus after GCI as a potential mechanism to limit neuronal cell death.MethodsGCI was induced by four-vessel occlusion in ovariectomized female SD rats. Specific agonist G1 or antagonist G36 of GPER was administrated using minipump, and antisense oligonucleotide (AS) of interleukin-1β receptor antagonist (IL1RA) was administrated using brain infusion kit. Protein expression of IL1RA, NF-κB-P65, phosphorylation of CREB (p-CREB), Bcl2, cleaved caspase 3, and microglial markers Iba1, CD11b, as well as inflammasome components NLRP3, ASC, cleaved caspase 1, and Cle-IL1β in the hippocampal CA1 region were investigated by immunofluorescent staining and Western blot analysis. The Duolink II in situ proximity ligation assay (PLA) was performed to detect the interaction between NLRP3 and ASC. Immunofluorescent staining for NeuN and TUNEL analysis were used to analyze neuronal survival and apoptosis, respectively. We performed Barnes maze and Novel object tests to compare the cognitive function of the rats.ResultsThe results showed that G1 attenuated GCI-induced elevation of Iba1 and CD11b in the hippocampal CA1 region at 14 days of reperfusion, and this effect was blocked by G36. G1 treatment also markedly decreased expression of the NLRP3-ASC-caspase 1 inflammasome and IL1β activation, as well as downstream NF-κB signaling, the effects reversed by G36 administration. Intriguingly, G1 caused a robust elevation in neurons of a well-known endogenous anti-inflammatory factor IL1RA, which was reversed by G36 treatment. G1 also enhanced p-CREB level in the hippocampus, a transcription factor known to enhance expression of IL1RA. Finally, in vivo IL1RA-AS abolished the anti-inflammatory, neuroprotective, and anti-apoptotic effects of G1 after GCI and reversed the cognitive-enhancing effects of G1 at 14 days after GCI.ConclusionsTaken together, the current results suggest that GPER preserves cognitive function following GCI in part by exerting anti-inflammatory effects and enhancing the defense mechanism of neurons by upregulating IL1RA.

Inhibition enhances spatially-specific calcium encoding of synaptic input patterns in a biologically constrained model.

Synaptic plasticity, which underlies learning and memory, depends on calcium elevation in neurons, but the precise relationship between calcium and spatiotemporal patterns of synaptic inputs is unclear. Here, we develop a biologically realistic computational model of striatal spiny projection neurons with sophisticated calcium dynamics, based on data from rodents of both sexes, to investigate how spatiotemporally clustered and distributed excitatory and inhibitory inputs affect spine calcium. We demonstrate that coordinated excitatory synaptic inputs evoke enhanced calcium elevation specific to stimulated spines, with lower but physiologically relevant calcium elevation in nearby non-stimulated spines. Results further show a novel and important function of inhibition-to enhance the difference in calcium between stimulated and non-stimulated spines. These findings suggest that spine calcium dynamics encode synaptic input patterns and may serve as a signal for both stimulus-specific potentiation and heterosynaptic depression, maintaining balanced activity in a dendritic branch while inducing pattern-specific plasticity.

Indomethacin preconditioning induces ischemic tolerance by modifying zinc availability in the brain

Intracellular zinc overload causes neuronal injury during the course of neurological disorders, whereas mild levels of zinc are beneficial to neurons. Previous reports indicated that non-steroidal anti-inflammatory drugs, including indomethacin and aspirin, can reduce the risk of ischemic stroke. This study found that chronic pretreatment of rats with indomethacin, a non-selective cyclooxygenase inhibitor, provided tolerance to ischemic injuries in an animal model of stroke by eliciting moderate zinc elevation in neurons. Consecutive intraperitoneal injection of indomethacin (3mg/kg/day for 28days) led to modest increases in intraneuronal zinc as well as synaptic zinc content, with no significant stimulation of neuronal death. Furthermore, indomethacin induced the expressions of intracellular zinc homeostatic and neuroprotective proteins, rendering the brain resistant against ischemic damages and improving neurological outcomes. However, administration of a zinc-chelator, N,N,N′,N′-tetra(2-picolyl)ethylenediamine (TPEN; 15mg/kg/day), immediately after indomethacin administration eliminated the beneficial actions of the drug. Therefore, indomethacin preconditioning can modulate intracellular zinc availability, contributing to ischemic tolerance in the brain after stroke.

Brain Insulin Resistance in Alzheimer's Disease and its Potential Treatment with GLP-1 Analogs

The prevalence of Alzheimer's disease is increasing rapidly in the absence of truly effective therapies. A promising strategy for developing such therapies is the treatment of brain insulin resistance, a common and early feature of Alzheimer's disease, closely tied to cognitive decline and capable of promoting many biological abnormalities in the disorder. The proximal cause of brain insulin resistance appears to be neuronal elevation in the serine phosphorylation of IRS-1, most likely due to amyloid-β-triggered microglial release of proinflammatory cytokines. Preclinically, the first line of defense is behavior-lowering peripheral insulin resistance (e.g., physical exercise and a Mediterranean diet supplemented with foods rich in flavonoids, curcumin and ω-3 fatty acids). More potent remediation is required, however, at clinical stages. Fortunately, the US FDA-approved antidiabetics exenatide (Byetta; Amylin Pharmaceuticals, Inc., CA, USA) and liraglutide (Victoza; Novo Nordisk A/S, Bagsvaerd, Denmark) are showing much promise in reducing Alzheimer's disease pathology and in restoring normal brain insulin responsiveness and cognitive function.

Distinct Activation Properties of the Nuclear Factor of Activated T-cells (NFAT) Isoforms NFATc3 and NFATc4 in Neurons

The Ca(2+)/calcineurin-dependent transcription factor NFAT (nuclear factor of activated T-cells) is implicated in regulating dendritic and axonal development, synaptogenesis, and neuronal survival. Despite the increasing appreciation for the importance of NFAT-dependent transcription in the nervous system, the regulation and function of specific NFAT isoforms in neurons are poorly understood. Here, we compare the activation of NFATc3 and NFATc4 in hippocampal and dorsal root ganglion neurons following electrically evoked elevations of intracellular Ca(2+) concentration ([Ca(2+)](i)). We find that NFATc3 undergoes rapid dephosphorylation and nuclear translocation that are essentially complete within 20 min, although NFATc4 remains phosphorylated and localized to the cytosol, only exhibiting nuclear localization following prolonged (1-3 h) depolarization. Knocking down NFATc3, but not NFATc4, strongly diminished NFAT-mediated transcription induced by mild depolarization in neurons. By analyzing NFATc3/NFATc4 chimeras, we find that the region containing the serine-rich region-1 (SRR1) mildly affects initial NFAT translocation, although the region containing the serine-proline repeats is critical for determining the magnitude of NFAT activation and nuclear localization upon depolarization. Knockdown of glycogen synthase kinase 3β (GSK3β) significantly increased the depolarization-induced nuclear localization of NFATc4. In contrast, inhibition of p38 or mammalian target of rapamycin (mTOR) kinases had no significant effect on nuclear import of NFATc4. Thus, electrically evoked [Ca(2+)](i) elevation in neurons rapidly and strongly activates NFATc3, whereas activation of NFATc4 requires a coincident increase in [Ca(2+)](i) and suppression of GSK3β, with differences in the serine-proline-containing region giving rise to these distinct activation properties of NFATc3 and NFATc4.

A new experimental model of focal seizures in the entorhinal cortex

Despite intensive studies, our understanding of the cellular and molecular mechanisms underlying epileptogenesis remains largely unsatisfactory. Our defective knowledge derives in part from the lack of adequate experimental models of the distinct phases that characterize the epileptic event, that is, initiation, propagation, and cessation. The aim of our study is the development of a new brain slice model in which a focal seizure can be repetitively evoked at a precise and predictable site. Epileptiform activities were studied by fast Ca²(+) imaging coupled with simultaneous single and double patch-clamp or extracellular recordings from neurons of entorhinal cortex (EC) slices from Wistar rats and C57BL6J mice at postnatal days 13-17. In the presence of 4-aminopyridine (4-AP) and low Mg²(+) , activation of layer V-VI neurons by local N-methyl-d-aspartate (NMDA) applications evolved into an ictal discharge (ID) that propagated to the entire EC. NMDA-evoked IDs were similar to spontaneous events. IDs with similar pattern and duration could be repetitively triggered from the same site by successive NMDA stimulations. The high ID reproducibility is an important feature of the model that allowed testing of the effects of currently used antiepileptic drugs (AEDs) on initiation, propagation, and cessation of focal ictal events. By offering the unique opportunity to repetitively evoke an ID from the same restricted site, this model represents a powerful approach to study the cellular and molecular events at the basis of initiation, propagation, and cessation of focal seizures.

Sphingosylphosphocholine effects on cultured astrocytes reveal mechanisms potentially involved in neurotoxicity in Niemann‐Pick type A disease

Niemann-Pick type A is a disease characterized by the absence of a functional SMPD1 (acidic sphingomyelinase) gene and the abnormal accumulation of sphingomyelin. Under these conditions, also sphingosylphosphocholine (SPC, a sphingomyelin metabolite) accumulates in various tissues, including the brain, where it might act as a toxic stimulus, contributing to the appearance of the neurological symptoms. We studied the effects of SPC on astrocytic and neuronal cultures from rat. In particular, we investigated the possibility that SPC acts on astrocytes and that this represents the first step leading to neurodegeneration. Our results show that acute administration of SPC to astrocytes in culture promotes Ca2+ responses and a release of glutamate that, in turn, leads to cytosolic [Ca2+] elevation in neurons. We also show that chronic stimulation by SPC leads astrocytes to proliferate, but can also change their phenotype towards an activated state that might contribute to the inflammatory responses. Interestingly, upon acute SPC stimulation, activated astrocytes release more glutamate. In conclusion, we show that both chronic and acute exposure to SPC can constitute harmful signals that may have a role in the sequence of events leading to neurodegeneration.

Correlating retinal function and amino acid immunocytochemistry following post-mortem ischemia

We wanted to determine the characteristics associated with electrophysiological and neurochemical changes secondary to ischemic insult as well as correlate these electrophysiological and neurochemical changes. A Ganzfeld source was used to elicit electroretinograms in anesthetized adult Sprague-Dawley rats. Following baseline recordings, one eye was removed for control quantitative amino acid immunocytochemistry, and ischemic insult was induced by cervical dislocation. Following the induction of ischemia, a single electroretinogram signal was collected at 1, 2, 4, 6, 8, 16, 32 or 64 min, after which the eye was removed for immunocytochemistry. The post-receptoral b-wave was undetectable after 1 min post-ischemia, whereas phototransduction declined more gradually and persisted for up to 16 min post-mortem. Both phototransduction saturated amplitude and sensitivity decayed with a similar time course ( t c=3.06 (2.73, 3.48) versus 3.29 (2.61, 4.62) min). Significant elevation of amino acid neurotransmitter levels was not observed until 6 min post-mortem. Between 8 and 16 min post-ischemia, glutamate and GABA were significantly accumulated in neurons and Müller cells ( p<0.05). Beyond 16 min, the neurotransmitter elevation in neurons and Müller cells was relatively attenuated. Aspartate immunoreactivity was significantly elevated at 4 and 6 min post-ischemia in neurons, prior to a change in any other amino acid. Moreover, of the amino acids assessed the post-ischemic change in aspartate immunoreactivity showed the best correlation with phototransduction decay ( r 2=0.68). Our findings show that complete impairment of phototransduction coincides with the accumulation of amino acid neurotransmitter. The correlation of aspartate immunoreactivity and phototransduction provides evidence of heightened glutamate oxidation during ischemic insult.

Mitochondrial calcium accumulation following activation of vanilloid (VR1) receptors by capsaicin in dorsal root ganglion neurons

Stimulation of the vanilloid (capsaicin) receptor (VR1), currently viewed as a molecular integrator of chemical and physical noxious stimuli, evoked intracellular Ca 2+ transients in a capsaicin-sensitive subpopulation of rat dorsal root ganglion neurons. These were comprised of an initial fast rise (seconds) followed by a long-lasting intracellular Ca 2+ recovery (tens of minutes). The rate of intracellular Ca 2+ recovery was dependent on the magnitude of intracellular Ca 2+ transients. Opening of voltage-operated Ca 2+ channels in the same neurons by K + depolarization evoked intracellular Ca 2+ elevation of a similar amplitude and rate of rise; however, the recovery of intracellular Ca 2+ to the prestimulated level was significantly faster. A mitochondrial uncoupler (10 μM carbonyl cyanide m-chlorophenylhydrasone) was used to reveal the role of mitochondria in intracellular Ca 2+ buffering. Carbonyl cyanide m-chlorophenylhydrasone-evoked elevation in intracellular Ca 2+ was greater in neurons previously stimulated with capsaicin compared with KCl. Neither extracellular Ca 2+ nor ATP depletion influenced significantly the carbonyl cyanide m-chlorophenylhydrasone-sensitive intracellular Ca 2+ elevation in neurons loaded with Ca 2+ via vanilloid 1 receptor stimulation. The effects of carbonyl cyanide m-chlorophenylhydrasone suggest that the amount of Ca 2+ buffered by mitochondria is greater when extracellular Ca 2+ enters the neuron via the vanilloid 1 receptor channel than via voltage-operated Ca 2+ channels. The long duration of intracellular Ca 2+ i decline in neurons stimulated with capsaicin, which depends on the amount of Ca 2+ buffered by mitochondria, may reflect a specific mechanism of Ca 2+ buffering following activation the pain receptor VR1.

The use of c-fos as a metabolic marker in neuronal pathway tracing

The use of c-fos protein (Fos) immunocytochemistry as a metabolic marker for tracing neuroanatomical connections, seizure pathways and sites of action of neuroactive drugs is discussed in this report. Fos immunocytochemistry will be very useful for these purposes providing that a number of potential problems are recognized and controlled. These include the observations that Fos exists basally in neurons and can be non-specifically elevated after behavioural stress; neuronal bursting is required to elevate Fos in neurons in anaesthetized animals; drugs such as ketamine can block Fos elevation in neurons: the time-course of Fos induction and decay varies with different inducing stimuli and the brain region sampled; and some brain regions do not express Fos after any treatments tried so far. To overcome these potential problems we list a number of steps that should be followed when using Fos immunocytochemistry as a metabolic marker of brain activity.