Role of sulfiredoxin as a peroxiredoxin-2 denitrosylase in human iPSC-derived dopaminergic neurons.

Recent studies have pointed to protein S-nitrosylation as a critical regulator of cellular redox homeostasis. For example, S-nitrosylation of peroxiredoxin-2 (Prx2), a peroxidase widely expressed in mammalian neurons, inhibits both enzymatic activity and protective function against oxidative stress. Here, using in vitro and in vivo approaches, we identify a role and reaction mechanism of the reductase sulfiredoxin (Srxn1) as an enzyme that denitrosylates (thus removing -SNO) from Prx2 in an ATP-dependent manner. Accordingly, by decreasing S-nitrosylated Prx2 (SNO-Prx2), overexpression of Srxn1 protects dopaminergic neural cells and human-induced pluripotent stem cell (hiPSC)-derived neurons from NO-induced hypersensitivity to oxidative stress. The pathophysiological relevance of this observation is suggested by our finding that SNO-Prx2 is dramatically increased in murine and human Parkinson's disease (PD) brains. Our findings therefore suggest that Srxn1 may represent a therapeutic target for neurodegenerative disorders such as PD that involve nitrosative/oxidative stress.

Isogenic human iPSC Parkinson's model shows nitrosative stress-induced dysfunction in MEF2-PGC1α transcription.

Parkinson's disease (PD) is characterized by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). An association has been reported between PD and exposure to mitochondrial toxins, including environmental pesticides paraquat, maneb, and rotenone. Here, using a robust, patient-derived stem cell model of PD allowing comparison of A53T α-synuclein (α-syn) mutant cells and isogenic mutation-corrected controls, we identify mitochondrial toxin-induced perturbations in A53T α-syn A9 DA neurons (hNs). We report a pathway whereby basal and toxin-induced nitrosative/oxidative stress results in S-nitrosylation of transcription factor MEF2C in A53T hNs compared to corrected controls. This redox reaction inhibits the MEF2C-PGC1α transcriptional network, contributing to mitochondrial dysfunction and apoptotic cell death. Our data provide mechanistic insight into gene-environmental interaction (GxE) in the pathogenesis of PD. Furthermore, using small-molecule high-throughput screening, we identify the MEF2C-PGC1α pathway as a therapeutic target to combat PD.

S-Nitrosylation of parkin as a novel regulator of p53-mediated neuronal cell death in sporadic Parkinson's disease.

BACKGROUND: Mutations in the gene encoding parkin, a neuroprotective protein with dual functions as an E3 ubiquitin ligase and transcriptional repressor of p53, are linked to familial forms of Parkinson's disease (PD). We hypothesized that oxidative posttranslational modification of parkin by environmental toxins may contribute to sporadic PD. RESULTS: We first demonstrated that S-nitrosylation of parkin decreased its activity as a repressor of p53 gene expression, leading to upregulation of p53. Chromatin immunoprecipitation as well as gel-shift assays showed that parkin bound to the p53 promoter, and this binding was inhibited by S-nitrosylation of parkin. Additionally, nitrosative stress induced apoptosis in cells expressing parkin, and this death was, at least in part, dependent upon p53. In primary mesencephalic cultures, pesticide-induced apoptosis was prevented by inhibition of nitric oxide synthase (NOS). In a mouse model of pesticide-induced PD, both S-nitrosylated (SNO-)parkin and p53 protein levels were increased, while administration of a NOS inhibitor mitigated neuronal death in these mice. Moreover, the levels of SNO-parkin and p53 were simultaneously elevated in postmortem human PD brain compared to controls. CONCLUSIONS: Taken together, our data indicate that S-nitrosylation of parkin, leading to p53-mediated neuronal cell death, contributes to the pathophysiology of sporadic PD.

Aberrant protein s-nitrosylation in neurodegenerative diseases.

S-Nitrosylation is a redox-mediated posttranslational modification that regulates protein function via covalent reaction of nitric oxide (NO)-related species with a cysteine thiol group on the target protein. Under physiological conditions, S-nitrosylation can be an important modulator of signal transduction pathways, akin to phosphorylation. However, with aging or environmental toxins that generate excessive NO, aberrant S-nitrosylation reactions can occur and affect protein misfolding, mitochondrial fragmentation, synaptic function, apoptosis or autophagy. Here, we discuss how aberrantly S-nitrosylated proteins (SNO-proteins) play a crucial role in the pathogenesis of neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. Insight into the pathophysiological role of aberrant S-nitrosylation pathways will enhance our understanding of molecular mechanisms leading to neurodegenerative diseases and point to potential therapeutic interventions.

S-nitrosylated SHP-2 contributes to NMDA receptor-mediated excitotoxicity in acute ischemic stroke.

Overproduction of nitric oxide (NO) can cause neuronal damage, contributing to the pathogenesis of several neurodegenerative diseases and stroke (i.e., focal cerebral ischemia). NO can mediate neurotoxic effects at least in part via protein S-nitrosylation, a reaction that covalently attaches NO to a cysteine thiol (or thiolate anion) to form an S-nitrosothiol. Recently, the tyrosine phosphatase Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) and its downstream pathways have emerged as important mediators of cell survival. Here we report that in neurons and brain tissue NO can S-nitrosylate SHP-2 at its active site cysteine, forming S-nitrosylated SHP-2 (SNO-SHP-2). We found that NMDA exposure in vitro and transient focal cerebral ischemia in vivo resulted in increased levels of SNO-SHP-2. S-Nitrosylation of SHP-2 inhibited its phosphatase activity, blocking downstream activation of the neuroprotective physiological ERK1/2 pathway, thus increasing susceptibility to NMDA receptor-mediated excitotoxicity. These findings suggest that formation of SNO-SHP-2 represents a key chemical reaction contributing to excitotoxic damage in stroke and potentially other neurological disorders.

Redox Regulation of Protein Function via Cysteine S-Nitrosylation and Its Relevance to Neurodegenerative Diseases.

Debilitating neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), can be attributed to neuronal cell damage in specific brain regions. An important hallmark of these diseases is increased oxidative and nitrosative stress that occurs via overproduction of highly reactive free radicals known as reactive oxygen species (ROS) and reactive nitrogen species (RNS). These molecules are normally removed by cellular antioxidant systems. Under physiological conditions, ROS/RNS are present at low levels, mediating several neurotrophic and neuroprotective signaling pathways. In contrast, under pathological conditions, there is a pronounced increase in ROS/RNS generation, impairing normal neurological function. Nitric oxide (NO) is one such molecule that functions as a signaling agent under physiological conditions but causes nitrosative stress under pathological conditions due to its enhanced production. As first reported by our group and colleagues, the toxic effects of NO can be in part attributed to thiol S-nitrosylation, a posttranslational modification of cysteine residues on specific proteins. Here, we review several reports appearing over the past decade showing that S-nitrosylation of an increasing number of proteins compromises important cellular functions, including mitochondrial dynamics, endoplasmic reticulum (ER) protein folding, and signal transduction, thereby promoting synaptic damage, cell death, and neurodegeneration.

Dual neuroprotective pathways of a pro-electrophilic compound via HSF-1-activated heat-shock proteins and Nrf2-activated phase 2 antioxidant response enzymes.

Activation of the Keap1/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway and consequent induction of phase 2 antioxidant enzymes is known to afford neuroprotection. Here, we present a series of novel electrophilic compounds that protect neurons via this pathway. Natural products, such as carnosic acid (CA), are present in high amounts in the herbs rosemary and sage as ortho-dihydroquinones, and have attracted particular attention because they are converted by oxidative stress to their active form (ortho-quinone species) that stimulate the Keap1/Nrf2 transcriptional pathway. Once activated, this pathway leads to the production of a series of antioxidant phase 2 enzymes. Thus, such dihydroquinones function as redox-activated 'pro-electrophiles'. Here, we explored the concept that related para-dihydroquinones represent even more effective bioactive pro-electrophiles for the induction of phase 2 enzymes without producing toxic side effects. We synthesized several novel para-hydroquinone-type pro-electrophilic compounds (designated D1 and D2) to analyze their protective mechanism. DNA microarray, PCR, and western blot analyses showed that compound D1 induced expression of heat-shock proteins (HSPs), including HSP70, HSP27, and DnaJ, in addition to phase 2 enzymes such as hemeoxygenase-1 (HO-1), NADP(H) quinine-oxidoreductase1, and the Na(+)-independent cystine/glutamate exchanger (xCT). Treatment with D1 resulted in activation of Nrf2 and heat-shock transcription factor-1 (HSF-1) transcriptional elements, thus inducing phase 2 enzymes and HSPs, respectively. In this manner, D1 protected neuronal cells from both oxidative and endoplasmic reticulum (ER)-related stress. Additionally, D1 suppressed induction of 78 kDa glucose-regulated protein (GRP78), an ER chaperone protein, and inhibited hyperoxidation of peroxiredoxin 2 (PRX2), a molecule that is in its reduced state can protect from oxidative stress. These results suggest that D1 is a novel pro-electrophilic compound that activates both the Nrf2 and HSF-1 pathways, and may thus offer protection from oxidative and ER stress.

NOS antagonism using viral vectors as an experimental strategy: implications for in vivo studies of cardiovascular control and peripheral neuropathies.

Nitric oxide, a free gaseous signalling molecule, has attracted the attention of numerous biologists and has been implicated in the regulation of the cardiovascular, nervous and immune system. However, the cellular mechanisms mediating nitric oxide modulation remain unclear. Upregulation by gene over-expression or down-regulation by gene inactivation of nitric oxide synthase has generated quantitative changes in abundance thereby permitting functional insights. We have tested and proved that genetic nitric oxide synthase antagonism using viral vectors, particularly with dominant negative mutants and microRNA 30-based short hairpin RNA, is an efficient and effective experimental approach to manipulate nitric oxide synthase expression both in vitro and in vivo.

NO orchestrates the loss of synaptic boutons from adult "sick" motoneurons: modeling a molecular mechanism.

Synapse elimination is the main factor responsible for the cognitive decline accompanying many of the neuropathological conditions affecting humans. Synaptic stripping of motoneurons is also a common hallmark of several motor pathologies. Therefore, knowledge of the molecular basis underlying this plastic process is of central interest for the development of new therapeutic tools. Recent advances from our group highlight the role of nitric oxide (NO) as a key molecule triggering synapse loss in two models of motor pathologies. De novo expression of the neuronal isoform of NO synthase (nNOS) in motoneurons commonly occurs in response to the physical injury of a motor nerve and in the course of amyotrophic lateral sclerosis. In both conditions, this event precedes synaptic withdrawal from motoneurons. Strikingly, nNOS-synthesized NO is "necessary" and "sufficient" to induce synaptic detachment from motoneurons. The mechanism involves a paracrine/retrograde action of NO on pre-synaptic structures, initiating a downstream signaling cascade that includes sequential activation of (1) soluble guanylyl cyclase, (2) cyclic guanosine monophosphate-dependent protein kinase, and (3) RhoA/Rho kinase (ROCK) signaling. Finally, ROCK activation promotes phosphorylation of regulatory myosin light chain, which leads to myosin activation and actomyosin contraction. This latter event presumably contributes to the contractile force to produce ending axon retraction. Several findings support that this mechanism may operate in the most prevalent neurodegenerative diseases.

Reduction in the motoneuron inhibitory/excitatory synaptic ratio in an early-symptomatic mouse model of amyotrophic lateral sclerosis.

Excitotoxicity is a widely studied mechanism underlying motoneuron degeneration in amyotrophic lateral sclerosis (ALS). Synaptic alterations that produce an imbalance in the ratio of inhibitory/excitatory synapses are expected to promote or protect against motoneuron excitotoxicity. In ALS patients, motoneurons suffer a reduction in their synaptic coverage, as in the transition from the presymptomatic (2-month-old) to early-symptomatic (3-month-old) stage of the hSOD1(G93A) mouse model of familial ALS. Net synapse loss resulted from inhibitory bouton loss and excitatory synapse gain. Furthermore, in 3-month-old transgenic mice, remaining inhibitory but not excitatory boutons attached to motoneurons showed reduction in the active zone length and in the spatial density of synaptic vesicles in the releasable pool near the active zone. Bouton degeneration/loss seems to be mediated by bouton vacuolization and by mechanical displacement due to swelling vacuolated dendrites. In addition, chronic treatment with a nitric oxide (NO) synthase inhibitor avoided inhibitory loss but not excitatory gain. These results indicate that NO mediates inhibitory loss occurring from the pre- to early-symptomatic stage of hSOD1(G93A) mice. This work contributes new insights on ALS pathogenesis, recognizing synaptic re-arrangement onto motoneurons as a mechanism favoring disease progression rather than as a protective homeostatic response against excitotoxic events.

Transgenic neuronal nitric oxide synthase expression induces axotomy-like changes in adult motoneurons.

Dysregulation of protein expression, function and/or aggregation is a hallmark of a number of neuropathological conditions. Among them, upregulation and/or de novo expression of the neuronal isoform of nitric oxide (NO) synthase (nNOS) commonly occurs in diverse neurodegenerative diseases and in axotomized motoneurons. We used adenoviral (AVV) and lentiviral (LVV) vectors to study the effects of de novo nNOS expression on the functional properties and synaptic array of motoneurons. AVV-nNOS injection into the genioglossus muscle retrogradely transduced neonatal hypoglossal motoneurons (HMNs). Ratiometric real-time NO imaging confirmed that transduced HMNs generated NO gradients in brain parenchyma (space constant: 12.3 µm) in response to a glutamatergic stimulus. Unilateral AVV-nNOS microinjection in the hypoglossal nucleus of adult rats induced axotomy-like changes in HMNs. Specifically, we found alterations in axonal conduction properties and the recruitment order of motor units and reductions in responsiveness to synaptic drive and in the linear density of synaptophysin-positive puncta opposed to HMN somata. Functional alterations were fully prevented by chronic treatment with nNOS or soluble guanylyl cyclase inhibitors. Synaptic and functional changes were also completely avoided by prior intranuclear injection of a neuron-specific LVV system for miRNA-mediated nNOS knock-down (LVV-miR-shRNA/nNOS). Furthermore, synaptic and several functional changes evoked by XIIth nerve injury were to a large extent prevented by intranuclear administration of LVV-miR-shRNA/nNOS. We suggest that nNOS up-regulation creates a repulsive NO gradient for synaptic boutons underlying most of the functional impairment undergone by injured motoneurons. This further strengthens the case for nNOS targeting as a plausible strategy for treatment of peripheral neuropathies and neurodegenerative disorders.

Evidence for endothelial nitric oxide as a negative regulator of Schwann cell dedifferentiation after peripheral nerve injury.

The loss of intimate contact with axons triggers Schwann cells (SCs) to switch from a myelin-producing phenotype to a dedifferentiated, proliferating non-myelin-forming state after nerve injury. SC dedifferentiation is required for effective nerve regeneration. Negative regulators of SC dedifferentiation are promising targets to accelerate function recovery in acquired peripheral neuropathies. We recently reported that nitric oxide (NO) synthesized by endothelial NO synthase (eNOS) slows down functional recovery and axon regeneration after XIIth nerve crushing. This harmful action could be effected by a NO-delaying action on SC dedifferentiation. Adenoviral vectors directing the expression of a dominant negative mutant for eNOS (AVV-TeNOS) or the enhanced green fluorescent protein (AVV-eGFP) were individually injected into the distal stump just after XIIth nerve crushing. Growth-associated protein 43 (GAP-43), strongly over-expressed in dedifferentiated SCs and regenerating axons, was up-regulated in AVV-TeNOS-transduced nerves relative to AVV-eGFP-treated nerves. AVV-TeNOS increased the number of GAP-43-positive cells and bands of Bungner but did not alter the number of Hoechst-positive nuclei relative to AVV-eGFP. These results signal endothelial NO as a negative regulator of the SC dedifferentiation process, but not of SC proliferation rate, after nerve injury. Vascular-derived factors should be taken into account as feasible extrinsic regulators of SC plasticity.

Nitric oxide induces pathological synapse loss by a protein kinase G-, Rho kinase-dependent mechanism preceded by myosin light chain phosphorylation.

The molecular signaling that underpins synapse loss in neuropathological conditions remains unknown. Concomitant upregulation of the neuronal nitric oxide (NO) synthase (nNOS) in neurodegenerative processes places NO at the center of attention. We found that de novo nNOS expression was sufficient to induce synapse loss from motoneurons at adult and neonatal stages. In brainstem slices obtained from neonatal animals, this effect required prolonged activation of the soluble guanylyl cyclase (sGC)/protein kinase G (PKG) pathway and RhoA/Rho kinase (ROCK) signaling. Synapse elimination involved paracrine/retrograde action of NO. Furthermore, before bouton detachment, NO increased synapse myosin light chain phosphorylation (p-MLC), which is known to trigger actomyosin contraction and neurite retraction. NO-induced MLC phosphorylation was dependent on cGMP/PKG-ROCK signaling. In adulthood, motor nerve injury induced NO/cGMP-dependent synaptic stripping, strongly affecting ROCK-expressing synapses, and increased the percentage of p-MLC-expressing inputs before synapse destabilization. We propose that this molecular cascade could trigger synapse loss underlying early cognitive/motor deficits in several neuropathological states.

Nitric-oxide-directed synaptic remodeling in the adult mammal CNS.

In adult mammals, learning, memory, and restoration of sensorimotor lost functions imply synaptic reorganization that requires diffusible messengers-mediated communication between presynaptic and postsynaptic structures. A candidate molecule to accomplish this function is the gaseous intercellular messenger nitric oxide (NO), which is involved in synaptogenesis and projection refinement during development; however, the role of NO in synaptic reorganization processes in adulthood remains to be established. In this work, we tested the hypothesis that this free radical is a mediator in the adult mammal CNS synaptic remodeling processes using a model of hypoglossal axonal injury recently developed by us. Axonal injury-induced disconnection of motoneurons from myocytes produces withdrawal of synaptic inputs to motoneurons and concomitant upregulation of the neuronal isoform of NO synthase (NOS-I). After recovery of the neuromuscular function, synaptic coverage is reestablished and NOS-I is downregulated. We also report, by using functional and morphological approaches, that chronic inhibition of the NO/cGMP pathway prevents synaptic withdrawal evoked by axon injury, despite the persistent muscle disconnection. After successful withdrawal of synaptic boutons, inhibition of NO synthesis, but not of cGMP, accelerated the recovery of synaptic coverage, although neuromuscular disconnection was maintained. Furthermore, protein S-nitrosylation was upregulated after nerve injury, and this effect was reversed by NOS-I inhibition. Our results suggest that during synaptic remodeling in the adult CNS, NO acts as a signal for synaptic detachment and inhibits synapse formation by cGMP-dependent and probably S-nitrosylation-mediated mechanisms, respectively. We also suggest a feasible role of NO in neurological disorders coursing with NOS-I upregulation.

Nerve injury reduces responses of hypoglossal motoneurones to baseline and chemoreceptor-modulated inspiratory drive in the adult rat.

The effects of peripheral nerve lesions on the membrane and synaptic properties of motoneurones have been extensively studied. However, minimal information exists about how these alterations finally influence discharge activity and motor output under physiological afferent drive. The aim of this work was to evaluate the effect of hypoglossal (XIIth) nerve crushing on hypoglossal motoneurone (HMN) discharge in response to the basal inspiratory afferent drive and its chemosensory modulation by CO(2). The evolution of the lesion was assessed by recording the compound muscle action potential evoked by XIIth nerve stimulation, which was lost on crushing and then recovered gradually to control values from the second to fourth weeks post-lesion. Basal inspiratory activities recorded 7 days post-injury in the nerve proximal to the lesion site, and in the nucleus, were reduced by 51.6% and 35.8%, respectively. Single unit antidromic latencies were lengthened by lesion, and unusually high stimulation intensities were frequently required to elicit antidromic spikes. Likewise, inspiratory modulation of unitary discharge under conditions in which chemoreceptor drive was varied by altering end-tidal CO(2) was reduced by more than 60%. Although the general recruitment scheme was preserved after XIIth nerve lesion, we noticed an increased proportion of low-threshold units and a reduced recruitment gain across the physiological range. Immunohistochemical staining of synaptophysin in the hypoglossal nuclei revealed significant reductions of this synaptic marker after nerve injury. Morphological and functional alterations recovered with muscle re-innervation. Thus, we report here that nerve lesion induced changes in the basal activity and discharge modulation of HMNs, concurrent with the loss of afferent inputs. Nevertheless, we suggest that an increase in membrane excitability, reported by others, and in the proportion of low-threshold units, could serve to preserve minimal electrical activity, prevent degeneration and favour axonal regeneration.