Degrading Seizures (or, Playing Tag with Nedd4-2)

Commentary

Epilepsy-Associated Gene Nedd4-2 Mediates Neuronal Activity and Seizure Susceptibility Through AMPA Receptors.

Zhu J, Lee KY, Jewett KA, Man HY, Chung HJ, Tsai NP. PLoS Genet 2017;13:e1006634.

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 a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, (AMPAR). Using a mouse model, termed Nedd4-2^andi, in which one of the major forms of Nedd4-2 in the brain is selectively deficient, we found that the spontaneous neuronal activity in Nedd4-2^andi 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 Nedd4-2^andi 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 hyper-activity and seizures. Our findings provide critical information to the future development of therapeutic strategies for patients who carry mutations of Nedd4-2.

Tightly regulated protein homeostasis is essential for neuronal function. In response to external stimuli, proteins are constantly added to and removed from synapses. The dynamic regulation of synaptic and neuronal protein content is achieved by protein synthesis, trafficking, and degradation. Defects in these processes have been associated with numerous brain disorders, including epilepsy. For many years, the major attention in epilepsy research has been on mechanisms that regulate the synthesis of proteins, such as mRNA transcription and translation. More recently, mechanisms that regulate the targeted removal of proteins—for example, through the ubiquitin proteasome system (UPS)—have become a focus of interest. Increased seizure susceptibility has been linked to gene mutations or aberrant expression of enzymes mediating protein ubiquitination (1–4), which tags proteins for their degradation by the proteasome (5).

However, little is known about the specific proteins and mechanisms that are affected by a dysfunctional UPS in epilepsy. The study by Zhu, Tsai, and colleagues is noteworthy because it is one of the first to shed light on the molecular mechanisms leading to heightened seizure susceptibility when a component of the UPS, the epilepsy-associated neural precursor cell expressed, developmentally down-regulated 4 (Nedd4-2), is impaired.

Nedd4-2 belongs to the large family of E3 ubiquitin ligases that are essential for ubiquitination. They covalently and target-specifically attach a 76-amino acid polypeptide called “ubiquitin” to certain lysines in proteins, providing a high degree of substrate diversity and specificity. Ubiquitin itself can be ubiquitinated, often giving rise to branched polyubiquitin chains. This polyubiquitination marks proteins for degradation by the proteasome, a multi-subunit complex that functions as the cell's internal disposal system for proteins. To complicate matters, ubiquitination is reversible through deubiquitinating enzymes, and not every polyubiquitinated protein will be degraded. Instead, ubiquitination can also label a protein for membrane internalization or target it to certain organelles (5).

Nedd4-2 has been discovered as an epilepsy susceptibility gene a few years ago, including photosensitive epilepsy and epileptic encephalopathies (3, 4), but the underlying mechanisms have been obscure. The study by Zhu et al. is an important advance because it is the first report of a protein substrate of Nedd4-2 that may contribute to the epilepsy phenotype when Nedd4-2 is mutated. Using pharmacologic and genetic tools, the authors provided several lines of evidence that the GluA1 subunit of the AMPA receptor, a known target of Nedd4-2 ubiquitination (6), is critical for Nedd4-2-associated neuronal hyperactivity.

For this study, Zhu et al. employed a mouse model of impaired Nedd4-2 function, Nedd4-2^andi, in which one of the major forms of Nedd4-2 in the brain is deficient. Multielectrode array and whole-cell patch-clamping recordings of cultured cortical neurons from Nedd4-2^andi mice confirmed previous work that loss of Nedd4 increases spontaneous neuronal activity and susceptibility to kainic acid-induced seizures (7). By pharmacologically manipulating AMPA receptors, the authors went on to demonstrate that Nedd4-2^andi neurons are more sensitive to AMPA receptor blockade than their wild-type counterparts but less susceptible to AMPA stimulation. This defect in AMPA receptor-mediated signaling was in line with previous studies showing that Nedd4-2 ubiquitinates the AMPA receptor subunit GluA1 and thereby restricts its cell surface expression (6). The significance of GluA1 for the Nedd4-2-associated epilepsy phenotype was supported twofold: First, the authors showed that genetic reduction of GluA1 decreased GluA1 cell surface expression to wild-type levels in vitro and reduced the elevated seizure susceptibility in Nedd4-2^andi mice. Secondly, they showed that Nedd4-2 harboring any one of three missense mutations found in humans with photosensitive epilepsy (3) failed to ubiquitinate GluA1, potentially due to impaired association with the adaptor protein 14-3-3. These latter findings are particularly relevant, because they provide the kind of mechanistic insight needed to fully understand the consequences of epilepsy-associated defects of the UPS.

The study by Zhu et al. corroborates that the well-balanced interplay of enzymes involved in protein ubiquitination is essential to prevent seizure-related hyperactivity and synchrony in the brain. Yet, as is common for scientific findings, their results generated more questions than they answered. It is, for example, uncertain if GluA1 reduction truly targets the underlying mechanism of Nedd4-2-associated seizure susceptibility. AMPA receptors are the main mediators of fast excitatory synaptic transmission; thus, it is not surprising that reducing their expression would decrease neuronal activity. Indeed, GluA1 heterozygosity also slightly reduced seizure susceptibility in Nedd4-2 wild-type animals, although no significant difference was detected in this relatively small group of animals. In vitro rescue experiments provided support for the relevance of GluA1 for the Nedd4-2-associated phenotype by showing that only recombinant wild-type Nedd4-2—but not the epilepsy-associated Nedd4-2 mutant versions—increased GluA1 surface expression and reduced spontaneous spike frequency in Nedd4-2-deficient neurons. Nonetheless, the hypothesis that additional substrates of Nedd4-2 may contribute to increased seizure susceptibility was supported by the observation that Nedd4-2 deficiency increased the amplitude as well as the frequency of miniature excitatory post-synaptic currents in cultured cortical neurons. This suggests pre- and postsynaptic defects and raises important questions: Which substrates of Nedd4-2 mediate the presynaptic defects? Do the epilepsy-associated mutations in Nedd4-2 affect ubiquitination of all its substrates? Is targeting one substrate of Nedd4-2 sufficient to reverse the epilepsy phenotype? It is important to note that the kainic acid seizure model may be biased towards a glutamate receptor mechanism. In the future, it will be interesting to assess if Nedd4-2 deficiency increases seizure susceptibility in other genetic or pharmacological epilepsy models, and if defects in Nedd4-2-mediated ubiquitination of GluA1 are a generalizable mechanism in epilepsy.

Despite these remaining questions, the findings by Zhu and colleagues provide valuable insight into the molecular mechanisms underlying Nedd4-2-associated epilepsy and therefore represent a critical first step towards linking epilepsy associated with a defect in the UPS to a potential mechanism-based therapy. To date, the FDA has approved only very few drugs targeting the UPS, e.g. the UPS-inhibitor Bortezomib for the use in cancer. However, improvements in screening tools and increasing knowledge about the molecular structure and function of the components of the UPS have led to a rise in drug discovery efforts and provide hope that more and improved UPS-targeting therapies will become available soon (8).

We are still at the beginning of understanding how defects in protein ubiquitination contribute to neuronal hyperactivity and epilepsy. This becomes even more evident in view of recent work, which offered a different perspective on the UPS and epilepsy: Engel et al. showed that kainic acid-induced seizures in mice lead to accumulation of polyubiquitinated proteins in the hippocampus, suggesting an “overload” of the system due to higher demand or impaired proteasome function (9). Thus, alterations in the UPS can be both consequence and cause of seizures. While studies like the one by Zhu and colleagues provide clear support for a causal function of impaired ubiquitination in epilepsy, more work is needed to delineate the role and regulation of the UPS and its components during acute seizures, epileptogenesis, and chronic epilepsy. To fully appreciate the defects caused by impaired UPS function in epilepsy, it will be essential to identify the proteins with altered ubiquitination and assess the resulting consequences on their activity, subcellular localization, and stability. Large-scale proteomic approaches and their validation, in addition to candidate approaches taken by Zhu et al., will be particularly informative. Uncovering the molecular mechanisms that regulate protein homeostasis in healthy and epileptic brains will ultimately facilitate the development of future therapeutic strategies.