Targeted protein S-nitrosylation of ACE2 inhibits SARS-CoV-2 infection.

Prevention of infection and propagation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a high priority in the Coronavirus Disease 2019 (COVID-19) pandemic. Here we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin-converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 spike protein, thereby inhibiting viral entry, infectivity and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and, thus, the spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model and, thus, provide a novel avenue to pursue therapy.

Targeted protein S-nitrosylation of ACE2 as potential treatment to prevent spread of SARS-CoV-2 infection.

Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and thus spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E-protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model, and thus provide a novel avenue for therapy.

TCA cycle metabolic compromise due to an aberrant S-nitrosoproteome in HIV-associated neurocognitive disorder with methamphetamine use.

In the brain, both HIV-1 and methamphetamine (meth) use result in increases in oxidative and nitrosative stress. This redox stress is thought to contribute to the pathogenesis of HIV-associated neurocognitive disorder (HAND) and further worsening cognitive activity in the setting of drug abuse. One consequence of such redox stress is aberrant protein S-nitrosylation, derived from nitric oxide, which may disrupt normal protein activity. Here, we report an improved, mass spectrometry-based technique to assess S-nitrosylated protein in human postmortem brains using selective enrichment of S-nitrosocysteine residues with an organomercury resin. The data show increasing S-nitrosylation of tricarboxylic acid (TCA) enzymes in the setting of HAND and HAND/meth use compared with HIV+ control brains without CNS pathology. The consequence is systematic inhibition of multiple TCA cycle enzymes, resulting in energy collapse that can contribute to the neuronal and synaptic damage observed in HAND and meth use.

Novel Therapeutic Approach for Excitatory/Inhibitory Imbalance in Neurodevelopmental and Neurodegenerative Diseases.

Excitatory/inhibitory (E/I) balance, defined as the balance between excitation and inhibition of synaptic activity in a neuronal network, accounts in part for the normal functioning of the brain, controlling, for example, normal spike rate. In many pathological conditions, this fine balance is perturbed, leading to excessive or diminished excitation relative to inhibition, termed E/I imbalance, reflected in network dysfunction. E/I imbalance has emerged as a contributor to neurological disorders that occur particularly at the extremes of life, including autism spectrum disorder and Alzheimer's disease, pointing to the vulnerability of neuronal networks at these critical life stages. Hence, it is important to develop approaches to rebalance neural networks. In this review, we describe emerging therapies that can normalize the E/I ratio or the underlying abnormality that contributes to the imbalance in electrical activity, thus improving neurological function in these maladies.

NitroSynapsin for the treatment of neurological manifestations of tuberous sclerosis complex in a rodent model.

Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by heterozygous mutations in the TSC1 or TSC2 gene. TSC is often associated with neurological, cognitive, and behavioral deficits. TSC patients also express co-morbidity with anxiety and mood disorders. The mechanism of pathogenesis in TSC is not entirely clear, but TSC-related neurological symptoms are accompanied by excessive glutamatergic activity and altered synaptic spine structures. To address whether extrasynaptic (e)NMDA-type glutamate receptor (NMDAR) antagonists, as opposed to antagonists that block physiological phasic synaptic activity, can ameliorate the synaptic and behavioral features of this disease, we utilized the Tsc2+/- mouse model of TSC to measure biochemical, electrophysiological, histological, and behavioral parameters in the mice. We found that antagonists that preferentially block tonic activity as found at eNMDARs, particularly the newer drug NitroSynapsin, provide biological and statistically significant improvement in Tsc2+/- phenotypes. Accompanying this improvement was correction of activity in the p38 MAPK-TSC-Rheb-mTORC1-S6K1 pathway. Deficits in hippocampal long-term potentiation (LTP), histological loss of synapses, and behavioral fear conditioning in Tsc2+/- mice were all improved after treatment with NitroSynapsin. Taken together, these results suggest that amelioration of excessive excitation, by limiting aberrant eNMDAR activity, may represent a novel treatment approach for TSC.

NitroSynapsin therapy for a mouse MEF2C haploinsufficiency model of human autism.

Transcription factor MEF2C regulates multiple genes linked to autism spectrum disorder (ASD), and human MEF2C haploinsufficiency results in ASD, intellectual disability, and epilepsy. However, molecular mechanisms underlying MEF2C haploinsufficiency syndrome remain poorly understood. Here we report that Mef2c +/-(Mef2c-het) mice exhibit behavioral deficits resembling those of human patients. Gene expression analyses on brains from these mice show changes in genes associated with neurogenesis, synapse formation, and neuronal cell death. Accordingly, Mef2c-het mice exhibit decreased neurogenesis, enhanced neuronal apoptosis, and an increased ratio of excitatory to inhibitory (E/I) neurotransmission. Importantly, neurobehavioral deficits, E/I imbalance, and histological damage are all ameliorated by treatment with NitroSynapsin, a new dual-action compound related to the FDA-approved drug memantine, representing an uncompetitive/fast off-rate antagonist of NMDA-type glutamate receptors. These results suggest that MEF2C haploinsufficiency leads to abnormal brain development, E/I imbalance, and neurobehavioral dysfunction, which may be mitigated by pharmacological intervention.

Molecular Pathway to Protection From Age-Dependent Photoreceptor Degeneration in Mef2 Deficiency.

Purpose: Photoreceptor degeneration in the retina is a major cause of blindness in humans. Elucidating mechanisms of degenerative and neuroprotective pathways in photoreceptors should afford identification and development of therapeutic strategies. Methods: We used mouse genetic models and improved methods for retinal explant cultures. Retinas were enucleated from Mef2d+/+ and Mef2d-/- mice, stained for MEF2 proteins and outer nuclear layer thickness, and assayed for apoptotic cells. Chromatin immunoprecipitation (ChIP) assays revealed MEF2 binding, and RT-qPCR showed levels of transcription factors. We used AAV2 and electroporation to express genes in retinal explants and electroretinograms to assess photoreceptor functionality. Results: We identify a prosurvival MEF2D-PGC1α pathway that plays a neuroprotective role in photoreceptors. We demonstrate that Mef2d-/- mouse retinas manifest decreased expression of PGC1α and increased photoreceptor cell loss, resulting in the absence of light responses. Molecular repletion of PGC1α protects Mef2d-/- photoreceptors and preserves light responsivity. Conclusions: These results suggest that the MEF2-PGC1α cascade may represent a new therapeutic target for drugs designed to protect photoreceptors from developmental- and age-dependent loss.

MEF2D haploinsufficiency downregulates the NRF2 pathway and renders photoreceptors susceptible to light-induced oxidative stress.

Gaining mechanistic insight into interaction between causative factors of complex multifactorial diseases involving photoreceptor damage might aid in devising effective therapies. Oxidative stress is one of the potential unifying mechanisms for interplay between genetic and environmental factors that contribute to photoreceptor pathology. Interestingly, the transcription factor myocyte enhancer factor 2d (MEF2D) is known to be important in photoreceptor survival, as knockout of this transcription factor results in loss of photoreceptors in mice. Here, using a mild light-induced retinal degeneration model, we show that the diminished MEF2D transcriptional activity in Mef2d+/- retina is further reduced under photostimulation-induced oxidative stress. Reactive oxygen species cause an aberrant redox modification on MEF2D, consequently inhibiting transcription of its downstream target, nuclear factor (erythroid-derived 2)-like 2 (NRF2). NRF2 is a master regulator of phase II antiinflammatory and antioxidant gene expression. In the Mef2d heterozygous mouse retina, NRF2 is not up-regulated to a normal degree in the face of light-induced oxidative stress, contributing to accelerated photoreceptor cell death. Furthermore, to combat this injury, we found that activation of the endogenous NRF2 pathway using proelectrophilic drugs rescues photoreceptors from photo-induced oxidative stress and may therefore represent a viable treatment for oxidative stress-induced photoreceptor degeneration, which is thought to contribute to some forms of retinitis pigmentosa and age-related macular degeneration.

Therapeutic advantage of pro-electrophilic drugs to activate the Nrf2/ARE pathway in Alzheimer's disease models.

Alzheimer's disease (AD) is characterized by synaptic and neuronal loss, which occurs at least partially through oxidative stress induced by oligomeric amyloid-ß (Aß)-peptide. Carnosic acid (CA), a chemical found in rosemary and sage, is a pro-electrophilic compound that is converted to its active form by oxidative stress. The active form stimulates the Keap1/Nrf2 transcriptional pathway and thus production of phase 2 antioxidant enzymes. We used both in vitro and in vivo models. For in vitro studies, we evaluated protective effects of CA on primary neurons exposed to oligomeric Aß. For in vivo studies, we used two transgenic mouse models of AD, human amyloid precursor protein (hAPP)-J20 mice and triple transgenic (3xTg AD) mice. We treated these mice trans-nasally with CA twice weekly for 3 months. Subsequently, we performed neurobehavioral tests and quantitative immunohistochemistry to assess effects on AD-related phenotypes, including learning and memory, and synaptic damage. In vitro, CA reduced dendritic spine loss in rat neurons exposed to oligomeric Aß. In vivo, CA treatment of hAPP-J20 mice improved learning and memory in the Morris water maze test. Histologically, CA increased dendritic and synaptic markers, and decreased astrogliosis, Aß plaque number, and phospho-tau staining in the hippocampus. We conclude that CA exhibits therapeutic benefits in rodent AD models and since the FDA has placed CA on the 'generally regarded as safe' (GRAS) list, thus obviating the need for safety studies, human clinical trials will be greatly expedited.

Levetiracetam inhibits oligomeric Aß-induced glutamate release from human astrocytes.

A recently identified mechanism for oligomeric Aß-induced glutamate release from astrocytes involves intracellular Ca elevation, potentially by Ca-dependent vesicular release. Evidence suggests that levetiracetam (LEV; Keppra), an antiepileptic drug, can improve cognitive performance in both humans with mild cognitive impairment and animal models of Alzheimer disease. Because LEV acts by modulating neurotransmitter release from neurons by interaction with synaptic vesicles, we tested the effect of LEV on Aß-induced astrocytic release of glutamate. We used a fluorescence resonance energy transfer-based glutamate sensor (termed SuperGluSnFR), whose structure is based on the ligand-binding site of glutamate receptors, to monitor glutamate release from primary cultures of human astrocytes exposed to oligomeric amyloid-ß peptide 1-42 (Aß42). We found that LEV (10 µM) inhibited oligomeric Aß-induced astrocytic glutamate release. In addition, we show that this Aß-induced glutamate release from astrocytes is sensitive to tetanus neurotoxin, an inhibitor of the vesicle release machinery. Taken together, our evidence suggests that LEV inhibits Aß-induced vesicular glutamate release from astrocytes and thus may underlie, at least in part, the ability of LEV to reduce hyperexcitability in Alzheimer disease.

Elevated glucose and oligomeric ß-amyloid disrupt synapses via a common pathway of aberrant protein S-nitrosylation.

Metabolic syndrome (MetS) and Type 2 diabetes mellitus (T2DM) increase risk for Alzheimer's disease (AD). The molecular mechanism for this association remains poorly defined. Here we report in human and rodent tissues that elevated glucose, as found in MetS/T2DM, and oligomeric ß-amyloid (Aß) peptide, thought to be a key mediator of AD, coordinately increase neuronal Ca(2+) and nitric oxide (NO) in an NMDA receptor-dependent manner. The increase in NO results in S-nitrosylation of insulin-degrading enzyme (IDE) and dynamin-related protein 1 (Drp1), thus inhibiting insulin and Aß catabolism as well as hyperactivating mitochondrial fission machinery. Consequent elevation in Aß levels and compromise in mitochondrial bioenergetics result in dysfunctional synaptic plasticity and synapse loss in cortical and hippocampal neurons. The NMDA receptor antagonist memantine attenuates these effects. Our studies show that redox-mediated posttranslational modification of brain proteins link Aß and hyperglycaemia to cognitive dysfunction in MetS/T2DM and AD.

Differential Effects of Pharmacologic and Genetic Modulation of NMDA Receptor Activity on HIV/gp120-Induced Neuronal Damage in an In Vivo Mouse Model.

HIV-associated neurocognitive disorder (HAND) consists of motor and cognitive dysfunction in a relatively large percentage of patients with AIDS. Prior work has suggested that at least part of the neuronal and synaptic damage observed in HAND may occur due to excessive stimulation of NMDA-type glutamate receptors (NMDARs). Here, we compared pharmacological and genetic manipulation of NMDAR activity using an improved derivative of the NMDAR antagonist memantine, termed NitroMemantine, and the modulatory NMDAR subunit GluN3A in the HIV/gp120 transgenic (tg) mouse model of HAND. Interestingly, we found that while both NitroMemantine and GluN3A have been shown to inhibit NMDAR activity, NitroMemantine protected synapses in gp120-tg mice, but overexpression of GluN3A augmented the damage. Given recent findings in the field, one explanation for this apparently paradoxical result is the location of the NMDARs primarily affected, with NitroMemantine inhibiting predominantly extrasynaptic pathologically activated NMDARs, but GluN3A disrupting normal NMDAR-mediated neuroprotective activity via inhibition of synaptic NMDARs.

Pharmacologically targeted NMDA receptor antagonism by NitroMemantine for cerebrovascular disease.

Stroke and vascular dementia are leading causes of morbidity and mortality. Neuroprotective therapies have been proposed but none have proven clinically tolerated and effective. While overstimulation of N-methyl-d-aspartate-type glutamate receptors (NMDARs) is thought to contribute to cerebrovascular insults, the importance of NMDARs in physiological function has made this target, at least in the view of many in 'Big Pharma,' 'undruggable' for this indication. Here, we describe novel NitroMemantine drugs, comprising an adamantane moiety that binds in the NMDAR-associated ion channel that is used to target a nitro group to redox-mediated regulatory sites on the receptor. The NitroMemantines are both well tolerated and effective against cerebral infarction in rodent models via a dual allosteric mechanism of open-channel block and NO/redox modulation of the receptor. Targeted S-nitrosylation of NMDARs by NitroMemantine is potentiated by hypoxia and thereby directed at ischemic neurons. Allosteric approaches to tune NMDAR activity may hold therapeutic potential for cerebrovascular disorders.

Nrf2 and HSF-1 Pathway Activation via Hydroquinone-Based Proelectrophilic Small Molecules is Regulated by Electrochemical Oxidation Potential.

Activation of the Kelch-like ECH-associated protein 1/nuclear factor (erythroid-derived 2)-like 2 and heat-shock protein 90/heat-shock factor-1 signal-transduction pathways plays a central role in combatting cellular oxidative damage and related endoplasmic reticulum stress. Electrophilic compounds have been shown to be activators of these transcription-mediated responses through S-alkylation of specific regulatory proteins. Previously, we reported that a prototype compound (D1, a small molecule representing a proelectrophilic, para-hydroquinone species) exhibited neuroprotective action by activating both of these pathways. We hypothesized that the para-hydroquinone moiety was critical for this activation because it enhanced transcription of these neuroprotective pathways to a greater degree than that of the corresponding ortho-hydroquinone isomer. This notion was based on the differential oxidation potentials of the isomers for the transformation of the hydroquinone to the active, electrophilic quinone species. Here, to further test this hypothesis, we synthesized a pair of para- and ortho-hydroquinone-based proelectrophilic compounds and measured their redox potentials using analytical cyclic voltammetry. The redox potential was then compared with functional biological activity, and the para-hydroquinones demonstrated a superior neuroprotective profile.

Aberrant protein S-nitrosylation contributes to the pathophysiology of neurodegenerative diseases.

Nitric oxide (NO) is a gasotransmitter that impacts fundamental aspects of neuronal function in large measure through S-nitrosylation, a redox reaction that occurs on regulatory cysteine thiol groups. For instance, S-nitrosylation regulates enzymatic activity of target proteins via inhibition of active site cysteine residues or via allosteric regulation of protein structure. During normal brain function, protein S-nitrosylation serves as an important cellular mechanism that modulates a diverse array of physiological processes, including transcriptional activity, synaptic plasticity, and neuronal survival. In contrast, emerging evidence suggests that aging and disease-linked environmental risk factors exacerbate nitrosative stress via excessive production of NO. Consequently, aberrant S-nitrosylation occurs and represents a common pathological feature that contributes to the onset and progression of multiple neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases. In the current review, we highlight recent key findings on aberrant protein S-nitrosylation showing that this reaction triggers protein misfolding, mitochondrial dysfunction, transcriptional dysregulation, synaptic damage, and neuronal injury. Specifically, we discuss the pathological consequences of S-nitrosylated parkin, myocyte enhancer factor 2 (MEF2), dynamin-related protein 1 (Drp1), protein disulfide isomerase (PDI), X-linked inhibitor of apoptosis protein (XIAP), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) under neurodegenerative conditions. We also speculate that intervention to prevent these aberrant S-nitrosylation events may produce novel therapeutic agents to combat neurodegenerative diseases.

Protection from cyanide-induced brain injury by the Nrf2 transcriptional activator carnosic acid.

Cyanide is a life-threatening, bioterrorist agent, preventing cellular respiration by inhibiting cytochrome c oxidase, resulting in cardiopulmonary failure, hypoxic brain injury, and death within minutes. However, even after treatment with various antidotes to protect cytochrome oxidase, cyanide intoxication in humans can induce a delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Additional mechanisms are thought to underlie cyanide-induced neuronal damage, including generation of reactive oxygen species. This may account for the fact that antioxidants prevent some aspects of cyanide-induced neuronal damage. Here, as a potential preemptive countermeasure against a bioterrorist attack with cyanide, we tested the CNS protective effect of carnosic acid (CA), a pro-electrophilic compound found in the herb rosemary. CA crosses the blood-brain barrier to up-regulate endogenous antioxidant enzymes via activation of the Nrf2 transcriptional pathway. We demonstrate that CA exerts neuroprotective effects on cyanide-induced brain damage in cultured rodent and human-induced pluripotent stem cell-derived neurons in vitro, and in vivo in various brain areas of a non-Swiss albino mouse model of cyanide poisoning that simulates damage observed in the human brain. Cyanide, a potential bioterrorist agent, can produce a chronic delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Here, cyanide poisoning treated with the proelectrophillic compound carnosic acid, results in reduced neuronal cell death in both in vitro and in vivo models through activation of the Nrf2/ARE transcriptional pathway. Carnosic acid is therefore a potential treatment for the toxic central nervous system (CNS) effects of cyanide poisoning. ARE, antioxidant responsive element; Nrf2 (NFE2L2, Nuclear factor (erythroid-derived 2)-like 2).

Transcriptional profiling of MEF2-regulated genes in human neural progenitor cells derived from embryonic stem cells.

[Briefly describe the contents of the Data in Brief article. Tell the reader the repository and reference number for the data in the abstract to.] The myocyte enhancer factor 2 (MEF2) family of transcription factors is highly expressed in the brain, and constitutes a key determinant of neuronal survival, differentiation, and synaptic plasticity. However, genome-wide transcriptional profiling of MEF2-regulated genes has not yet been fully elucidated, particularly at the neural stem cell stage. Here we report the results of microarray analysis comparing mRNAs isolated from human neural progenitor/stem cells (hNPCs) derived from embryonic stem cells expressing a control vector versus progenitors expressing a constitutively-active form of MEF2 (MEF2CA), which increases MEF2 activity. Microarray experiments were performed using the Illumina Human HT-12 V4.0 expression beadchip (GEO#: GSE57184). By comparing vector-control cells to MEF2CA cells, microarray analysis identified 1880 unique genes that were differentially expressed. Among these genes, 1121 genes were upregulated and 759 genes were down-regulated. Our results provide a valuable resource for identifying transcriptional targets of MEF2 in hNPCs.

S-nitrosylation-mediated redox transcriptional switch modulates neurogenesis and neuronal cell death.

Redox-mediated posttranslational modifications represent a molecular switch that controls major mechanisms of cell function. Nitric oxide (NO) can mediate redox reactions via S-nitrosylation, representing transfer of an NO group to a critical protein thiol. NO is known to modulate neurogenesis and neuronal survival in various brain regions in disparate neurodegenerative conditions. However, a unifying molecular mechanism linking these phenomena remains unknown. Here, we report that S-nitrosylation of myocyte enhancer factor 2 (MEF2) transcription factors acts as a redox switch to inhibit both neurogenesis and neuronal survival. Structure-based analysis reveals that MEF2 dimerization creates a pocket, facilitating S-nitrosylation at an evolutionally conserved cysteine residue in the DNA binding domain. S-Nitrosylation disrupts MEF2-DNA binding and transcriptional activity, leading to impaired neurogenesis and survival in vitro and in vivo. Our data define a molecular switch whereby redox-mediated posttranslational modification controls both neurogenesis and neurodegeneration via a single transcriptional signaling cascade.