Tmod2 Is a Regulator of Cocaine Responses through Control of Striatal and Cortical Excitability and Drug-Induced Plasticity.

Drugs of abuse induce neuroadaptations, including synaptic plasticity, that are critical for transition to addiction, and genes and pathways that regulate these neuroadaptations are potential therapeutic targets. Tropomodulin 2 (Tmod2) is an actin-regulating gene that plays an important role in synapse maturation and dendritic arborization and has been implicated in substance abuse and intellectual disability in humans. Here, we mine the KOMP2 data and find that Tmod2 knock-out mice show emotionality phenotypes that are predictive of addiction vulnerability. Detailed addiction phenotyping shows that Tmod2 deletion does not affect the acute locomotor response to cocaine administration. However, sensitized locomotor responses are highly attenuated in these knock-outs, indicating perturbed drug-induced plasticity. In addition, Tmod2 mutant animals do not self-administer cocaine indicating lack of hedonic responses to cocaine. Whole-brain MR imaging shows differences in brain volume across multiple regions, although transcriptomic experiments did not reveal perturbations in gene coexpression networks. Detailed electrophysiological characterization of Tmod2 KO neurons showed increased spontaneous firing rate of early postnatal and adult cortical and striatal neurons. Cocaine-induced synaptic plasticity that is critical for sensitization is either missing or reciprocal in Tmod2 KO nucleus accumbens shell medium spiny neurons, providing a mechanistic explanation of the cocaine response phenotypes. Combined, these data, collected from both males and females, provide compelling evidence that Tmod2 is a major regulator of plasticity in the mesolimbic system and regulates the reinforcing and addictive properties of cocaine.

Amyloid ß Proteoforms Elucidated by Quantitative LC/MS in the 5xFAD Mouse Model of Alzheimer's Disease.

Numerous Aß proteoforms, identified in the human brain, possess differential neurotoxic and aggregation propensities. These proteoforms contribute in unknown ways to the conformations and resultant pathogenicity of oligomers, protofibrils, and fibrils in Alzheimer's disease (AD) manifestation owing to the lack of molecular-level specificity to the exact chemical composition of underlying protein products with widespread interrogating techniques, like immunoassays. We evaluated Aß proteoform flux using quantitative top-down mass spectrometry (TDMS) in a well-studied 5xFAD mouse model of age-dependent Aß-amyloidosis. Though the brain-derived Aß proteoform landscape is largely occupied by Aß1-42, 25 different forms of Aß with differential solubility were identified. These proteoforms fall into three natural groups defined by hierarchical clustering of expression levels in the context of mouse age and proteoform solubility, with each group sharing physiochemical properties associated with either N/C-terminal truncations or both. Overall, the TDMS workflow outlined may hold tremendous potential for investigating proteoform-level relationships between insoluble fibrils and soluble Aß, including low-molecular-weight oligomers hypothesized to serve as the key drivers of neurotoxicity. Similarly, the workflow may also help to validate the utility of AD-relevant animal models to recapitulate amyloidosis mechanisms or possibly explain disconnects observed in therapeutic efficacy in animal models vs humans.

Infantile spasms-linked Nedd4-2 mediates hippocampal plasticity and learning via cofilin signaling.

Individuals affected by infantile spasms (IS), such as those carrying mutations in an IS-linked gene, neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4-2), exhibit developmental delays and learning disabilities, but the underlying mechanism is unknown. Using conditional Nedd4-2 knockout mice, we uncover that Nedd4-2 functions to maintain the excitatory synapses in hippocampal neurons and allows for late-phase long-term synaptic potentiation (L-LTP) at Schaffer collateral synapses in the hippocampus. We also find that Nedd4-2 is required for multiple forms of hippocampus-dependent learning and memory. Mechanistically, we show that loss of Nedd4-2 leads to a decrease in actin polymerization caused by reduced phosphorylation of the actin depolymerizing protein cofilin. A cell-permeable peptide promoting phosphorylation of endogenous cofilin in Nedd4-2 knockout neurons restores the number of hippocampal excitatory synapses and hippocampal L-LTP and partially restores hippocampus-dependent learning in mice. Taken together, our results reveal a novel mechanism underlying IS-associated learning disabilities and may provide information for future therapeutic strategies for IS.

E3 ubiquitin ligase Nedd4-2 exerts neuroprotective effects during endoplasmic reticulum stress.

The neural precursor cell expressed developmentally down-regulated protein 4-like (Nedd4-2) is an E3 ubiquitin ligase critical for neurodevelopment and homeostasis of neural circuit excitability. While dysregulation of Nedd4-2 has been linked to elevated seizure susceptibility through impaired ubiquitination of multiple direct substrates, it remains largely unclear whether Nedd4-2 interconnects other cellular pathways that affect neuronal activity and seizure susceptibility. Here, we first showed that Nedd4-2 associates with the endoplasmic reticulum (ER) and regulates the expression of multiple ER-resident proteins. Furthermore, utilizing Nedd4-2 conditional knockout mice, we showed that Nedd4-2 is required for the maintenance of spontaneous neural activity and excitatory synapses following the induction of ER stress. When analyzing activation of the canonical pathways of ER stress response, we found that Nedd4-2 is required for phosphorylation of eIF2α. While phosphorylation of eIF2α has been shown to reduce seizure susceptibility, attempts to facilitate phosphorylation of eIF2α in Nedd4-2 conditional knockout mice failed to produce such a beneficial function, suggesting a role for Nedd4-2 in integrating the ER stress response to modulate seizure susceptibility. Altogether, our study demonstrates neuroprotective functions of Nedd4-2 during ER stress in neurons and could provide insight into neurological diseases in which the expression or activity of Nedd4-2 is impaired.

Unbiased proteomic screening identifies a novel role for the E3 ubiquitin ligase Nedd4-2 in translational suppression during ER stress.

Endoplasmic reticulum (ER) stress occurs when protein folding or maturation is disrupted. A malfunction in the ER stress response can lead to cell death and has been observed in many neurological diseases. However, how the ER stress response is regulated in neuronal cells remains largely unclear. Here, we studied an E3 ubiquitin ligase named neural precursor cell expressed developmentally down-regulated protein 4-like (Nedd4-2). Nedd4-2 is highly expressed in the brain and has a high affinity toward ubiquitinating membrane-bound proteins. We first utilized unbiased proteomic profiling with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) of isolated membrane fractions from mouse whole brains to identify novel targets of Nedd4-2. Through this screen, we found that the expression and ubiquitination of ribosomal proteins are regulated by Nedd4-2 and we confirmed an association between Nedd4-2 and ribosomes through ribosome sedimentation and polysome profiling. Further, we utilized immunoprecipitation and western blotting to show that induction of ER stress promotes an association between Nedd4-2 and ribosomal proteins, which is mediated through dephosphorylation of Nedd4-2 at serine-342. This increased interaction between Nedd4-2 and ribosomal proteins in turn mediates ER stress-associated translational suppression. In summary, the results of this study demonstrate a novel regulatory mechanism underlying the ER stress response and a novel function of Nedd4-2 in translational control. Our findings may shed light on neurological diseases in which the ER stress response or the function of Nedd4-2 is dysregulated.

Epilepsy-associated gene Nedd4-2 mediates neuronal activity and seizure susceptibility through AMPA receptors.

The neural precursor cell expressed developmentally down-regulated gene 4-2, Nedd4-2, is an epilepsy-associated gene with at least three missense mutations identified in epileptic patients. Nedd4-2 encodes a ubiquitin E3 ligase that has high affinity toward binding and ubiquitinating membrane proteins. It is currently unknown how Nedd4-2 mediates neuronal circuit activity and how its dysfunction leads to seizures or epilepsies. In this study, we provide evidence to show that Nedd4-2 mediates neuronal activity and seizure susceptibility through ubiquitination of GluA1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, (AMPAR). Using a mouse model, termed Nedd4-2andi, in which one of the major forms of Nedd4-2 in the brain is selectively deficient, we found that the spontaneous neuronal activity in Nedd4-2andi cortical neuron cultures, measured by a multiunit extracellular electrophysiology system, was basally elevated, less responsive to AMPAR activation, and much more sensitive to AMPAR blockade when compared with wild-type cultures. When performing kainic acid-induced seizures in vivo, we showed that elevated seizure susceptibility in Nedd4-2andi mice was normalized when GluA1 is genetically reduced. Furthermore, when studying epilepsy-associated missense mutations of Nedd4-2, we found that all three mutations disrupt the ubiquitination of GluA1 and fail to reduce surface GluA1 and spontaneous neuronal activity when compared with wild-type Nedd4-2. Collectively, our data suggest that impaired GluA1 ubiquitination contributes to Nedd4-2-dependent neuronal hyperactivity and seizures. Our findings provide critical information to the future development of therapeutic strategies for patients who carry mutations of Nedd4-2.

C2-lacking isoform of Nedd4-2 regulates excitatory synaptic strength through GluA1 ubiquitination-independent mechanisms.

Neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4-2) is an epilepsy-associated gene, which encodes a ubiquitin E3 ligase that is highly expressed in the brain. Nedd4-2's substrates include many ion channels and receptors because its N-terminal C2 domain guides Nedd4-2 to the cell membrane. We previously found that Nedd4-2 ubiquitinates the glutamate receptor subunit 1 (GluA1) subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, which leads to reduction of neuronal excitability and seizures in mice. However, despite awareness of a Nedd4-2 isoform with no C2 domain, the functions of this isoform remain elusive. In this study, we showed that the C2-lacking Nedd4-2 has reduced membrane distribution and exhibits reduced affinity toward ubiquitinating GluA1. However, when expressed in primary cortical neurons, we found that the C2-lacking Nedd4-2 exhibits a similar activity toward reducing excitatory synaptic strength as does the C2-containing Nedd4-2. In an attempt to identify novel Nedd4-2 substrates that could mediate excitatory synaptic strength, we used unbiased proteomic screening and found multiple synaptic regulators that were up-regulated in the brain of conditional Nedd4-2 knockout mice, including protein phosphatase 3 catalytic subunit-α (PPP3CA; alternately called calcineurin A-α). We confirmed PPP3CA as a substrate of the C2-lacking Nedd4-2 and showed that all three epilepsy-associated missense mutations of Nedd4-2 disrupted PPP3CA ubiquitination. Altogether, our results revealed novel potential Nedd4-2 substrates and suggest that the C2-lacking Nedd4-2 represses excitatory synaptic strength most likely through GluA1 ubiquitination-independent mechanisms. These findings provide novel information to further our knowledge about Nedd4-2-dependent neuronal excitability homeostasis and pathological hyperexcitability when Nedd4-2 is compromised.

Structural characterization of the DNA-binding mechanism underlying the copper(II)-sensing MarR transcriptional regulator.

Multiple antibiotic resistance regulator (MarR) family proteins are widely conserved transcription factors that control bacterial resistance to antibiotics, environmental stresses, as well as the regulation of virulence determinants. Escherichia coli MarR, the prototype member of this family, has recently been shown to undergo copper(II)-catalyzed inter-dimer disulfide bond formation via a unique cysteine residue (Cys80) residing in its DNA-binding domain. However, despite extensive structural characterization of the MarR family proteins, the structural mechanism for DNA binding of this copper(II)-sensing MarR factor remains elusive. Here, we report the crystal structures of DNA-bound forms of MarR, which revealed a unique, concerted generation of two new helix-loop-helix motifs that facilitated MarR's DNA binding. Structural analysis and electrophoretic mobility shift assays (EMSA) show that the flexibility of Gly116 in the center of helix α5 and the extensive hydrogen-bonding interactions at the N-terminus of helix α1 together assist the reorientation of the wHTH domains and stabilize MarR's DNA-bound conformation.

The multiple antibiotic resistance regulator MarR is a copper sensor in Escherichia coli.

The widely conserved multiple antibiotic resistance regulator (MarR) family of transcription factors modulates bacterial detoxification in response to diverse antibiotics, toxic chemicals or both. The natural inducer for Escherichia coli MarR, the prototypical transcription repressor within this family, remains unknown. Here we show that copper signaling potentiates MarR derepression in E. coli. Copper(II) oxidizes a cysteine residue (Cys80) on MarR to generate disulfide bonds between two MarR dimers, thereby inducing tetramer formation and the dissociation of MarR from its cognate promoter DNA. We further discovered that salicylate, a putative MarR inducer, and the clinically important bactericidal antibiotics norfloxacin and ampicillin all stimulate intracellular copper elevation, most likely through oxidative impairment of copper-dependent envelope proteins, including NADH dehydrogenase-2. This membrane-associated copper oxidation and liberation process derepresses MarR, causing increased bacterial antibiotic resistance. Our study reveals that this bacterial transcription regulator senses copper(II) as a natural signal to cope with stress caused by antibiotics or the environment.

The tumor suppressor p53 guides GluA1 homeostasis through Nedd4-2 during chronic elevation of neuronal activity.

Chronic activity perturbation in neurons can trigger homeostatic mechanisms to restore the baseline function. Although the importance and dysregulation of neuronal activity homeostasis has been implicated in neurological disorders such as epilepsy, the complete signaling by which chronic changes in neuronal activity initiate the homeostatic mechanisms is unclear. We report here that the tumor suppressor p53 and its signaling are involved in neuronal activity homeostasis. Upon chronic elevation of neuronal activity in primary cortical neuron cultures, the ubiquitin E3 ligase, murine double minute- 2 (Mdm2), is phosphorylated by the kinase Akt. Phosphorylated Mdm2 triggers the degradation of p53 and subsequent induction of a p53 target gene, neural precursor cell expressed developmentally down-regulated gene 4-like (Nedd4-2). Nedd4-2 encodes another ubiquitin E3 ligase. We identified glutamate receptor subunit 1 (GluA1), subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors as a novel substrate of Nedd4-2. The regulation of GluA1 level is known to be crucial for neuronal activity homeostasis. We confirmed that, by pharmacologically inhibiting Mdm2-mediated p53 degradation or genetically reducing Nedd4-2 in a mouse model, the GluA1 ubiquitination and down-regulation induced by chronically elevated neuronal activity are both attenuated. Our findings demonstrate the first direct function of p53 in neuronal homeostasis and elucidate a new mechanism by which cortical neurons respond to chronic activity perturbation.

Ubiquitination and E3 Ubiquitin Ligases in Rare Neurological Diseases with Comorbid Epilepsy.

Ubiquitination is a post-translational modification that can dynamically alter the function, degradation and transport of a protein, as well as its interaction with other proteins, and activity of an enzyme. Dysfunctional ubiquitination is detrimental to normal cellular functions, and can result in severe diseases. Over the last decade, although much research has focused on deciphering the role of the ubiquitination/ubiquitin proteasome system (UPS) in the onset and progression of various neurological disorders, the specific relationship between ubiquitination and various epilepsies has not been carefully reviewed. As an increasing amount of research has revealed the roles of ubiquitination in the trafficking of ion channels and the turn-over of synaptic receptors, it is crucial to take a deeper look into ubiquitination-associated epilepsy. Here, we review the role of ubiquitination in maintaining normal cellular activities in neurons and recent findings on the dysregulation of ubiquitination in epilepsy. We particularly focus on rare neurological disorders with comorbid epilepsy in the hope of drawing more attention to this area. Through categorizing epilepsy-associated E3 ubiquitin ligases and their substrates and discussing ubiquitination-related rare neurological disorders, we summarize where the field stands at the moment and what directions we should consider in the future.

Feedback modulation of neural network synchrony and seizure susceptibility by Mdm2-p53-Nedd4-2 signaling.

BACKGROUND: Neural network synchrony is a critical factor in regulating information transmission through the nervous system. Improperly regulated neural network synchrony is implicated in pathophysiological conditions such as epilepsy. Despite the awareness of its importance, the molecular signaling underlying the regulation of neural network synchrony, especially after stimulation, remains largely unknown. RESULTS: In this study, we show that elevation of neuronal activity by the GABA(A) receptor antagonist, Picrotoxin, increases neural network synchrony in primary mouse cortical neuron cultures. The elevation of neuronal activity triggers Mdm2-dependent degradation of the tumor suppressor p53. We show here that blocking the degradation of p53 further enhances Picrotoxin-induced neural network synchrony, while promoting the inhibition of p53 with a p53 inhibitor reduces Picrotoxin-induced neural network synchrony. These data suggest that Mdm2-p53 signaling mediates a feedback mechanism to fine-tune neural network synchrony after activity stimulation. Furthermore, genetically reducing the expression of a direct target gene of p53, Nedd4-2, elevates neural network synchrony basally and occludes the effect of Picrotoxin. Finally, using a kainic acid-induced seizure model in mice, we show that alterations of Mdm2-p53-Nedd4-2 signaling affect seizure susceptibility. CONCLUSION: Together, our findings elucidate a critical role of Mdm2-p53-Nedd4-2 signaling underlying the regulation of neural network synchrony and seizure susceptibility and reveal potential therapeutic targets for hyperexcitability-associated neurological disorders.