Non-canonical Keap1-independent activation of Nrf2 in astrocytes by mild oxidative stress.

The transcription factor Nrf2 is a stress-responsive master regulator of antioxidant, detoxification and proteostasis genes. In astrocytes, Nrf2-dependent gene expression drives cell-autonomous cytoprotection and also non-cell-autonomous protection of nearby neurons, and can ameliorate pathology in several acute and chronic neurological disorders associated with oxidative stress. However, the value of astrocytic Nrf2 as a therapeutic target depends in part on whether Nrf2 activation by disease-associated oxidative stress occludes the effect of any Nrf2-activating drug. Nrf2 activation classically involves the inhibition of interactions between Nrf2's Neh2 domain and Keap1, which directs Nrf2 degradation. Keap1 inhibition is mediated by the modification of cysteine residues on Keap1, and can be triggered by electrophilic small molecules such as tBHQ. Here we show that astrocytic Nrf2 activation by oxidative stress involves Keap1-independent non-canonical signaling. Keap1 deficiency elevates basal Nrf2 target gene expression in astrocytes and occludes the effects of tBHQ, oxidative stress still induced strong Nrf2-dependent gene expression in Keap1-deficient astrocytes. Moreover, while tBHQ prevented protein degradation mediated via Nrf2's Neh2 domain, oxidative stress did not, consistent with a Keap1-independent mechanism. Moreover the effects of oxidative stress and tBHQ on Nrf2 target gene expression are additive, not occlusive. Mechanistically, oxidative stress enhances the transactivation potential of Nrf2's Neh5 domain in a manner dependent on its Cys-191 residue. Thus, astrocytic Nrf2 activation by oxidative stress involves Keap1-independent non-canonical signaling, meaning that further Nrf2 activation by Keap1-inhibiting drugs may be a viable therapeutic strategy.

Corrigendum: Synaptic NMDA receptor activity is coupled to the transcriptional control of the glutathione system.

This corrects the article DOI: 10.1038/ncomms7761.

Expression of mRNA Encoding Mcu and Other Mitochondrial Calcium Regulatory Genes Depends on Cell Type, Neuronal Subtype, and Ca2+ Signaling.

Uptake of Ca2+ into the mitochondrial matrix controls cellular metabolism and survival-death pathways. Several genes are implicated in controlling mitochondrial Ca2+ uptake (mitochondrial calcium regulatory genes, MCRGs), however, less is known about the factors which influence their expression level. Here we have compared MCRG mRNA expression, in neural cells of differing type (cortical neurons vs. astrocytes), differing neuronal subtype (CA3 vs. CA1 hippocampus) and in response to Ca2+ influx, using a combination of qPCR and RNA-seq analysis. Of note, we find that the Mcu-regulating Micu gene family profile differs substantially between neurons and astrocytes, while expression of Mcu itself is markedly different between CA3 and CA1 regions in the adult hippocampus. Moreover, dynamic control of MCRG mRNA expression in response to membrane depolarization-induced Ca2+ influx is also apparent, resulting in repression of Letm1, as well as Mcu. Thus, the mRNA expression profile of MCRGs is not fixed, which may cause differences in the coupling between cytoplasmic and mitochondrial Ca2+, as well as diversity of mitochondrial Ca2+ uptake mechanisms.

Neuronal development is promoted by weakened intrinsic antioxidant defences due to epigenetic repression of Nrf2.

Forebrain neurons have weak intrinsic antioxidant defences compared with astrocytes, but the molecular basis and purpose of this is poorly understood. We show that early in mouse cortical neuronal development in vitro and in vivo, expression of the master-regulator of antioxidant genes, transcription factor NF-E2-related-factor-2 (Nrf2), is repressed by epigenetic inactivation of its promoter. Consequently, in contrast to astrocytes or young neurons, maturing neurons possess negligible Nrf2-dependent antioxidant defences, and exhibit no transcriptional responses to Nrf2 activators, or to ablation of Nrf2's inhibitor Keap1. Neuronal Nrf2 inactivation seems to be required for proper development: in maturing neurons, ectopic Nrf2 expression inhibits neurite outgrowth and aborization, and electrophysiological maturation, including synaptogenesis. These defects arise because Nrf2 activity buffers neuronal redox status, inhibiting maturation processes dependent on redox-sensitive JNK and Wnt pathways. Thus, developmental epigenetic Nrf2 repression weakens neuronal antioxidant defences but is necessary to create an environment that supports neuronal development.

Synaptic NMDA receptor activity is coupled to the transcriptional control of the glutathione system.

How the brain's antioxidant defenses adapt to changing demand is incompletely understood. Here we show that synaptic activity is coupled, via the NMDA receptor (NMDAR), to control of the glutathione antioxidant system. This tunes antioxidant capacity to reflect the elevated needs of an active neuron, guards against future increased demand and maintains redox balance in the brain. This control is mediated via a programme of gene expression changes that boosts the synthesis, recycling and utilization of glutathione, facilitating ROS detoxification and preventing Puma-dependent neuronal apoptosis. Of particular importance to the developing brain is the direct NMDAR-dependent transcriptional control of glutathione biosynthesis, disruption of which can lead to degeneration. Notably, these activity-dependent cell-autonomous mechanisms were found to cooperate with non-cell-autonomous Nrf2-driven support from astrocytes to maintain neuronal GSH levels in the face of oxidative insults. Thus, developmental NMDAR hypofunction and glutathione system deficits, separately implicated in several neurodevelopmental disorders, are mechanistically linked.

Insight into a neuron's preferential susceptibility to oxidative stress.

Neurons are more vulnerable to oxidative stress than astrocytes, the reasons for which have yet to be fully elucidated. Understanding the cellular and molecular mechanisms which contribute to this enhanced vulnerability is key to efforts aimed at ameliorating neuronal health and resilience to oxidative stress, particularly in the context of neurodegenerative disease, which is characterized by progressive dysfunction and loss of neurons specifically, and in which oxidative stress is considered a central aetiological contributor. Biological factors which may influence neuronal susceptibility to oxidative stress, in normal and neurodegenerative contexts, are reviewed in the present article, with a focus on properties intrinsic to the neuronal cell type and on properties related to neuronal reliance on surrounding astrocytes.

Calmodulin kinase IV-dependent CREB activation is required for neuroprotection via NMDA receptor-PSD95 disruption.

NMDA-type glutamate receptors mediate both trophic and excitotoxic signalling in CNS neurons. We have previously shown that blocking NMDAR- post-synaptic density-95 (PSD95) interactions provides significant protection from excitotoxicity and in vivo ischaemia; however, the mechanism of neuroprotection is unclear. Here, we report that blocking PSD-95 interactions with the Tat-NR2B9c peptide enhances a Ca²âº-dependent protective pathway converging on cAMP Response Element binding protein (CREB) activation. We provide evidence that Tat-NR2B9c neuroprotection from oxygen glucose deprivation and NMDA toxicity occurs in parallel with the activation of calmodulin kinase signalling and is dependent on a sustained phosphorylation of the CREB transcription factor and its activator CaMKIV. Tat-NR2B9c-dependent neuroprotection and CREB phosphorylation are blocked by coapplication of CaM kinase (KN93 and STO-609) or CREB (KG-501) inhibitors, and by siRNA knockdown of CaMKIV. These results are mirrored in vivo in a rat model of permanent focal ischaemia. Tat-NR2B9c application significantly reduces infarct size and causes a selective and sustained elevation in CaMKIV phosphorylation; effects which are blocked by coadministration of KN93. Thus, calcium-dependent nuclear signalling via CaMKIV and CREB is critical for neuroprotection via NMDAR-PSD95 blockade, both in vitro and in vivo. This study highlights the importance of maintaining neuronal function following ischaemic injury. Future stroke research should target neurotrophic and pro-survival signal pathways in the development of novel neuroprotective strategies.

The subtype of GluN2 C-terminal domain determines the response to excitotoxic insults.

It is currently unclear whether the GluN2 subtype influences NMDA receptor (NMDAR) excitotoxicity. We report that the toxicity of NMDAR-mediated Ca(2+) influx is differentially controlled by the cytoplasmic C-terminal domains of GluN2B (CTD(2B)) and GluN2A (CTD(2A)). Studying the effects of acute expression of GluN2A/2B-based chimeric subunits with reciprocal exchanges of their CTDs revealed that CTD(2B) enhances NMDAR toxicity, compared to CTD(2A). Furthermore, the vulnerability of forebrain neurons in vitro and in vivo to NMDAR-dependent Ca(2+) influx is lowered by replacing the CTD of GluN2B with that of GluN2A by targeted exon exchange in a mouse knockin model. Mechanistically, CTD(2B) exhibits stronger physical/functional coupling to the PSD-95-nNOS pathway, which suppresses protective CREB activation. Dependence of NMDAR excitotoxicity on the GluN2 CTD subtype can be overcome by inducing high levels of NMDAR activity. Thus, the identity (2A versus 2B) of the GluN2 CTD controls the toxicity dose-response to episodes of NMDAR activity.

Activation of Nrf2-regulated glutathione pathway genes by ischemic preconditioning.

Prophylactic pharmacological activation of astrocytic gene expression driven by the transcription factor Nrf2 boosts antioxidant defences and protects against neuronal loss in ischemia and other disease models. However, the role of Nrf2 in mediating endogenous neuroprotective responses is less clear. We recently showed that Nrf2 is activated by mild oxidative stress in both rodent and human astrocytes. Moreover, brief exposure to ischemic conditions was found to activate Nrf2 both in vivo and in vitro, and this was found to contribute to neuroprotective ischemic preconditioning. Here we show that transient ischemic conditions in vitro and in vivo cause an increase in the expression of Nrf2 target genes associated with the glutathione pathway, including those involved in glutathione biosynthesis and cystine uptake. Taken together, these studies indicate that astrocytic Nrf2 may represent an important mediator of endogenous neuroprotective preconditioning pathways.

The influence of synaptic activity on neuronal health.

According to the theory of neuronal health, neurons exist in a spectrum of states ranging from highly resilient to vulnerable. An unhealthy neuron may be rendered dysfunctional or non-viable by an insult that would ordinarily be non-toxic to a healthy neuron. Over the years it has become clear that a neuron's health is subject to dynamic regulation by electrical or synaptic activity. This review highlights recently identified activity dependent signalling events that boost neuronal health through the transcriptional control of pro-apoptotic and anti-apoptotic genes, the enhancement of antioxidant defences, and the regulation of mitochondrial and neurotrophic factor availability. Furthermore, activity dependent signals have recently been shown to influence a variety of events specific to individual neurodegenerative diseases, which will also be highlighted.

CNS peroxiredoxins and their regulation in health and disease.

Oxidative stress is thought to be a contributing factor in many chronic neurodegenerative pathologies, as well as acute cerebrovascular disorders such as stroke. Peroxiredoxins are a family of antioxidant enzymes that reduce peroxides directly through the use of a redox active cysteine within their active site, which in the process becomes oxidized. In order to cycle back to the reduced state, many peroxiredoxins rely on thiol-dependent reduction by the ubiquitous antioxidant enzyme thioredoxin. Peroxiredoxins, together with thioredoxin and thioredoxin's own 'recycling enzyme', thioredoxin reductase, represent an antioxidant enzymic system of growing significance in the context of neuronal physiology and pathology. Overexpression, knockdown, and knockout approaches have demonstrated an important role for peroxiredoxins in protecting neurons from oxidative insults. It is also becoming clear that neuronal peroxiredoxins are subjected to post-translational modifications that impair function as part of disease pathology. Conversely, components of this pathway are also subject to dynamic upregulation such as via endogenous synaptic activity-dependent signaling and induction of the Nrf2-dependent Phase II response. As such, the thioredoxin-peroxiredoxin system represents a potential therapeutic target for central nervous system disorders associated with oxidative stress.

Neuronal activity controls the antagonistic balance between peroxisome proliferator-activated receptor-γ coactivator-1α and silencing mediator of retinoic acid and thyroid hormone receptors in regulating antioxidant defenses.

Transcriptional coactivators and corepressors often have multiple targets and can have opposing actions on transcription and downstream physiological events. The coactivator peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α is under-expressed in Huntington's disease and is a regulator of antioxidant defenses and mitochondrial biogenesis. We show that in primary cortical neurons, expression of PGC-1α strongly promotes resistance to excitotoxic and oxidative stress in a cell autonomous manner, whereas knockdown increases sensitivity. In contrast, the transcriptional corepressor silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) specifically antagonizes PGC-1α-mediated antioxidant effects. The antagonistic balance between PGC-1α and SMRT is upset in favor of PGC-1α by synaptic activity. Synaptic activity triggers nuclear export of SMRT reliant on multiple regions of the protein. Concomitantly, synaptic activity post-translationally enhances the transactivating potential of PGC-1α in a p38-dependent manner, as well as upregulating cyclic-AMP response element binding protein-dependent PGC-1α transcription. Activity-dependent targeting of PGC-1α results in enhanced gene expression mediated by the thyroid hormone receptor, a prototypical transcription factor coactivated by PGC-1α and repressed by SMRT. As a consequence of these events, SMRT is unable to antagonize PGC-1α-mediated resistance to oxidative stress in synaptically active neurons. Thus, PGC-1α and SMRT are antagonistic regulators of neuronal vulnerability to oxidative stress. Further, this coactivator-corepressor antagonism is regulated by the activity status of the cell, with implications for neuronal viability.

Suppression of the intrinsic apoptosis pathway by synaptic activity.

Synaptic activity promotes resistance to diverse apoptotic insults, the mechanism behind which is incompletely understood. We show here that a coordinated downregulation of core components of the intrinsic apoptosis pathway by neuronal activity forms a key part of the underlying mechanism. Activity-dependent protection against apoptotic insults is associated with inhibition of cytochrome c release in most but not all neurons, indicative of anti-apoptotic signaling both upstream and downstream of this step. We find that enhanced firing activity suppresses expression of the proapoptotic BH3-only member gene Puma in a NMDA receptor-dependent, p53-independent manner. Puma expression is sufficient to induce cytochrome c loss and neuronal apoptosis. Puma deficiency protects neurons against apoptosis and also occludes the protective effect of synaptic activity, while blockade of physiological NMDA receptor activity in the developing mouse brain induces neuronal apoptosis that is preceded by upregulation of Puma. However, enhanced activity can also confer resistance to Puma-induced apoptosis, acting downstream of cytochrome c release. This mechanism is mediated by transcriptional suppression of apoptosome components Apaf-1 and procaspase-9, and limiting caspase-9 activity, since overexpression of procaspase-9 accelerates the rate of apoptosis in active neurons back to control levels. Synaptic activity does not exert further significant anti-apoptotic effects downstream of caspase-9 activation, since an inducible form of caspase-9 overrides the protective effect of synaptic activity, despite activity-induced transcriptional suppression of caspase-3. Thus, suppression of apoptotic gene expression may synergize with other activity-dependent events such as enhancement of antioxidant defenses to promote neuronal survival.

Role of histone acetylation in the activity-dependent regulation of sulfiredoxin and sestrin 2.

Peroxiredoxins are neuroprotective antioxidant enzymes that reduce hydroperoxides and protect neurons against oxidative stress. However, they can be inactivated through hyperoxidation of their active site cysteine, an event that can take place in the brain in response to oxidative insults such as stroke and also normal aging. Synaptic activity promotes the reduction of hyperoxidized peroxiredoxins in neurons, and induces the expression of sulfiredoxin (Srxn1) and sestrin 2 (Sesn2) which have been reported to mediate this. We have investigated the importance of histone acetylation in the regulation of these genes, to understand more about how these genes are regulated by synaptic activity. We show that the sestrin 2 promoter undergoes activity-dependent histone acetylation, which contributes to its transcriptional activation. In contrast, promoter-proximal histone acetylation is not involved in the activity-dependent induction of sulfiredoxin. Nevertheless, expression of both sestrin 2 and sulfiredoxin can be induced by enhancing histone acetylation through treatment of neurons with the histone deacetylase inhibitor trichostatin A (TSA). Furthermore, protective doses of TSA inhibit the formation of hyperoxidized peroxiredoxins in neurons exposed to oxidative insults. Histone deacetylases are emerging therapeutic targets in neurodegenerative disorders associated with oxidative stress. Our results indicate that manipulating the histone acetylase-deacetylase balance in neurons may mimic the effects of synaptic activity in preventing the oxidative inactivation of peroxiredoxins.

ADAM-10 over-expression increases cortical synaptogenesis.

Cortical cholinergic, glutamatergic and GABAergic terminals become upregulated during early stages of the transgenic amyloid pathology. Abundant evidence suggests that sAPP alpha, the product of the non-amyloidogenic alpha-secretase pathway, is neurotrophic both in vitro and when exogenously applied in vivo. The disintegrin metalloprotease ADAM-10 has been shown to have alpha-secretase activity in vivo. To determine whether sAPP alpha has an endogenous biological influence on cortical presynaptic boutons in vivo, we quantified cortical cholinergic, glutamatergic and GABAergic presynaptic bouton densities in either ADAM-10 moderate expressing (ADAM-10 mo) transgenic mice, which moderately overexpress ADAM-10, or age-matched non-transgenic controls. Both early and late ontogenic time points were investigated. ADAM-10 mo transgenic mice display significantly elevated cortical cholinergic, glutamatergic and GABAergic presynaptic bouton densities at the early time point (8 months). Only the cholinergic presynaptic bouton density remains significantly elevated in late-staged ADAM-10 mo transgenic animals (18 months). To confirm that the observed elevations were due to increased levels of endogenous murine sAPP alpha, exogenous human sAPP alpha was infused into the cortex of non-transgenic control animals for 1 week. Exogenous infusion of sAPP alpha led to significant elevations in the cholinergic, glutamatergic and GABAergic cortical presynaptic bouton populations. These results are the first to demonstrate an in vivo influence of ADAM-10 on neurotransmitter-specific cortical synaptic plasticity and further confirm the neurotrophic influence of sAPP alpha on cortical synaptogenesis.

Paradoxical upregulation of glutamatergic presynaptic boutons during mild cognitive impairment.

Synaptic integrity is now recognized as a central component of Alzheimer's disease. Surprisingly, however, the structural status of glutamatergic synapses in Alzheimer's disease is unclear, despite the fact that glutamate is the major excitatory transmitter of the CNS and has key roles in excitotoxicity and long-term potentiation. The identification of specific markers of glutamatergic neurons now allows an assessment of the structural involvement of the glutamatergic system across progressive stages of the Alzheimer's pathology, an opportunity not afforded by previously used neurochemical approaches. Glutamatergic presynaptic bouton density and dystrophic neurite abundance were quantified in midfrontal gyrus brain tissue from subjects with no cognitive impairment, mild cognitive impairment, or mild- or severe-stage Alzheimer's disease. Our study demonstrates a striking pathology-dependent pattern of glutamatergic synaptic remodeling with disease progression. Subjects with mild cognitive impairment display a paradoxical elevation in glutamatergic presynaptic bouton density, a situation akin to that observed in the cholinergic system, which then depletes and drops with disease progression. This pattern of synaptic remodeling mirrors our previous findings in transgenic animal models and is of major relevance to current transmitter-based therapeutics.

Altered synaptic function in Alzheimer's disease.

Alzheimer's disease is the leading cause of dementia in the elderly, presenting itself clinically by progressive loss of memory and learning. Since synaptic density correlates more closely with cognitive impairment than any other pathological lesion observable in the disease pathology, an increased understanding of the mechanisms behind synaptic disconnection is of vital importance. Our lab investigated the neurotransmitter-specific status of distinct cortical presynaptic bouton populations in various transgenic mouse models of the Alzheimer's-like amyloid pathology in order to assess their involvement throughout the progression of the pathology. These studies have revealed that the amyloid pathology appears to progress in a neurotransmitter-specific manner where the cholinergic terminals appear most vulnerable, followed by the glutamatergic terminals and finally by the somewhat more resilient GABAergic terminals. This review will discuss additional studies which also provide evidence of a neurotransmitter-specific pathology as well as comment on the potential explanations for the observed vulnerabilities, touching upon metabolic demand, trophic support and receptor mediated activation.

The amyloid pathology progresses in a neurotransmitter-specific manner.

Past studies using transgenic models of early-staged amyloid pathology, have suggested that the amyloid pathology progresses in a neurotransmitter-specific manner where cholinergic terminals appear most vulnerable, followed by glutamatergic terminals and finally by somewhat more resistant GABAergic terminals. To determine whether this neurotransmitter-specific progression persists at later pathological stages, presynaptic bouton densities, and the areas of occupation and localization of plaque adjacent dystrophic neurites were quantified in 18-month-old APP(K670N, M671L)+PS1(M146L) doubly transgenic mice. Quantification revealed that transgenic animals had significantly lower cholinergic, glutamatergic and GABAergic presynaptic bouton densities. Cholinergic and glutamatergic dystrophic neurites appear to be heavily influenced by fibrillar Abeta as both types displayed a decreasing area of occupation with respect to increasing plaque size. Furthermore, cholinergic dystrophic neurites reside in closer proximity to plaques than glutamatergic dystrophic neurites, while GABAergic dystrophic neurites were minimal regardless of plaque size. To investigate whether similarities exist in the human AD pathology, a monoclonal antibody (McKA1) against the human vesicular glutamate transporter 1 (VGluT1) was developed. Subsequent staining in AD brain tissue revealed the novel presence of glutamatergic dystrophic neurites, to our knowledge the first evidence of a structural glutamatergic deficit in the AD pathology.