Amy Lin

Nedd4-mediated AMPA receptor ubiquitination regulates receptor turnover and trafficking

J. Neurochem. (2011) 119, 27–39.

Na,K-ATPase Activity Regulates AMPA Receptor Turnover through Proteasome-Mediated Proteolysis

Neuronal activity largely depends on two key components on the membrane: the Na,K-ATPase (NKA) that maintains the ion gradients and sets the foundation of excitability, and the ionotropic glutamatergic AMPA receptors (AMPARs) through which sodium influx forms the driving force for excitation. Because the frequent sodium transients from glutamate receptor activity need to be efficiently extruded, a functional coupling between NKA and AMPARs should be a necessary cellular device for synapse physiology. We show that NKA is enriched at synapses and associates with AMPARs. NKA dysfunction induces a rapid reduction in AMPAR cell-surface expression as well as total protein abundance, leading to a long-lasting depression in synaptic transmission. AMPAR proteolysis requires sodium influx, proteasomal activity and receptor internalization. These data elucidate a novel mechanism by which NKA regulates AMPAR turnover and thereby synaptic strength and brain function.

Design and Selection of Toca 511 for Clinical Use: Modified Retroviral Replicating Vector With Improved Stability and Gene Expression

Retroviral replicating vectors (RRVs) are a nonlytic alternative to oncolytic replicating viruses as anticancer agents, being selective both for dividing cells and for cells that have defects in innate immunity and interferon responsiveness. Tumor cells fit both these descriptions. Previous publications have described a prototype based on an amphotropic murine leukemia virus (MLV), encoding yeast cytosine deaminase (CD) that converts the prodrug 5-fluorocytosine (5-FC) to the potent anticancer drug, 5-fluorouracil (5-FU) in an infected tumor. We report here the selection of one lead clinical candidate based on a general design goal to optimize the genetic stability of the virus and the CD activity produced by the delivered transgene. Vectors were tested for titer, genetic stability, CD protein and enzyme activity, ability to confer susceptibility to 5-FC, and preliminary in vivo antitumor activity and stability. One vector, Toca 511, (aka T5.0002) encoding an optimized CD, shows a threefold increased specific activity in infected cells over infection with the prototype RRV and shows markedly higher genetic stability. Animal testing demonstrated that Toca 511 replicates stably in human tumor xenografts and, after 5-FC administration, causes complete regression of such xenografts. Toca 511 (vocimagene amiretrorepvec) has been taken forward to preclinical and clinical trials.

Ubiquitination of neurotransmitter receptors and postsynaptic scaffolding proteins.

The human brain is made up of an extensive network of neurons that communicate by forming specialized connections called synapses. The amount, location, and dynamic turnover of synaptic proteins, including neurotransmitter receptors and synaptic scaffolding molecules, are under complex regulation and play a crucial role in synaptic connectivity and plasticity, as well as in higher brain functions. An increasing number of studies have established ubiquitination and proteasome-mediated degradation as universal mechanisms in the control of synaptic protein homeostasis. In this paper, we focus on the role of the ubiquitin-proteasome system (UPS) in the turnover of major neurotransmitter receptors, including glutamatergic and nonglutamatergic receptors, as well as postsynaptic receptor-interacting proteins.

Changes in expression of c-Fos protein following cocaine-cue extinction learning.

Extinguishing abnormally strengthened learned responses to cues associated with drugs of abuse remains a key tactic for alleviating addiction. To assist in developing pharmacotherapies to augment exposure therapy for relapse prevention, investigation into neurobiological underpinnings of drug-cue extinction learning is needed. We used regional analyses of c-Fos and GluR2 protein expression to delineate neural activity and plasticity that may be associated with cocaine-cue extinction learning. Rats were trained to self-administer cocaine paired with a light cue, and later underwent a single 2h extinction session for which cocaine was withheld but response-contingent cues were presented (cocaine-cue extinction). Control groups consisted of rats yoked to animals self-administering cocaine and receiving saline non-contingently followed by an extinction session, or rats trained to self-administer cocaine followed by a no-extinction session for which levers were retracted, and cocaine and cues were withheld. Among 11 brain sites examined, extinction training increased c-Fos expression in basolateral amygdala and prelimbic prefrontal cortex of cocaine-cue extinguished rats relative to both control conditions. In dorsal subiculum and infralimbic prefrontal cortex, extinction training increased c-Fos expression in both cocaine-cue and saline-cue extinguished rats relative to the no-extinction control condition. GluR2 protein expression was not altered in any site examined after extinction or control training. Findings suggest that basolateral amygdala and prelimbic prefrontal cortex neurons are activated during acquisition of cocaine-cue extinction learning, a process that is independent of changes in GluR2 abundance. Other sites are implicated in processing the significance of cues that are present early in extinction training.

Dissociations in Cortical Morphometry in Youth with Down Syndrome: Evidence for Reduced Surface Area but Increased Thickness

Detailed descriptions of cortical anatomy in youth with Down syndrome (DS), the most common genetic cause of intellectual disability (ID), are scant. Thus, the current study examined deviations in cortical thickness (CT) and surface area (SA), at high spatial resolution, in youth with DS, to identify focal differences relative to typically developing (TD) youth. Participants included 31 youth with DS and 45 age- and sex-matched TD controls (mean age ∼16 years; range = 5-24 years). All participants completed T1-weighted ASSET-calibrated magnetization prepared rapid gradient echo scans on a 3-T magnetic resonance imaging scanner. Replicating prior investigations, cortical volume was reduced in DS compared with controls. However, a novel dissociation for SA and CT was found-namely, SA was reduced (predominantly in frontal and temporal regions) while CT was increased (notably in several regions thought to belong to the default mode network; DMN). These findings suggest that reductions in SA rather than CT are driving the cortical volume reductions reported in prior investigations of DS. Moreover, given the link between DMN functionality and Alzheimers symptomatology in chromosomally typical populations, future DS studies may benefit from focusing on the cortex in DMN regions, as such investigations may provide clues to the precocious onset of Alzheimers disease in this at-risk group.

Mapping the Stability of Human Brain Asymmetry across Five Sex-Chromosome Aneuploidies

The human brain displays stereotyped and early emerging patterns of cortical asymmetry in health. It is unclear if these asymmetries are highly sensitive to genetic and environmental variation or fundamental features of the brain that can survive severe developmental perturbations. To address this question, we mapped cortical thickness (CT) asymmetry in a group of genetically defined disorders known to impact CT development. Participants included 137 youth with one of five sex-chromosome aneuploidies [SCAs; XXX (n = 28), XXY (n = 58), XYY (n = 26), XXYY (n = 20), and XXXXY (n = 5)], and 169 age-matched typically developing controls (80 female). In controls, we replicated previously reported rightward inferior frontal and leftward lateral parietal CT asymmetry. These opposing frontoparietal CT asymmetries were broadly preserved in all five SCA groups. However, we also detected foci of shifting CT asymmetry with aneuploidy, which fell almost exclusively within regions of significant CT asymmetry in controls. Specifically, X-chromosome aneuploidy accentuated normative rightward inferior frontal asymmetries, while Y-chromosome aneuploidy reversed normative rightward medial prefrontal and lateral temporal asymmetries. These findings indicate that (1) the stereotyped normative pattern of opposing frontoparietal CT asymmetry arises from developmental mechanisms that can withstand gross chromosomal aneuploidy and (2) X and Y chromosomes can exert focal, nonoverlapping and directionally opposed influences on CT asymmetry within cortical regions of significant asymmetry in health. Our study attests to the resilience of developmental mechanisms that support the global patterning of CT asymmetry in humans, and motivates future research into the molecular bases and functional consequences of sex chromosome dosage effects on CT asymmetry.

Mapping 22q11.2 Gene Dosage Effects on Brain Morphometry

Reciprocal chromosomal rearrangements at the 22q11.2 locus are associated with elevated risk of neurodevelopmental disorders. The 22q11.2 deletion confers the highest known genetic risk for schizophrenia, but a duplication in the same region is strongly associated with autism and is less common in schizophrenia cases than in the general population. Here we conducted the first study of 22q11.2 gene dosage effects on brain structure in a sample of 143 human subjects: 66 with 22q11.2 deletions (22q-del; 32 males), 21 with 22q11.2 duplications (22q-dup; 14 males), and 56 age- and sex-matched controls (31 males). 22q11.2 gene dosage varied positively with intracranial volume, gray and white matter volume, and cortical surface area (deletion < control < duplication). In contrast, gene dosage varied negatively with mean cortical thickness (deletion > control > duplication). Widespread differences were observed for cortical surface area with more localized effects on cortical thickness. These diametric patterns extended into subcortical regions: 22q-dup carriers had a significantly larger right hippocampus, on average, but lower right caudate and corpus callosum volume, relative to 22q-del carriers. Novel subcortical shape analysis revealed greater radial distance (thickness) of the right amygdala and left thalamus, and localized increases and decreases in subregions of the caudate, putamen, and hippocampus in 22q-dup relative to 22q-del carriers. This study provides the first evidence that 22q11.2 is a genomic region associated with gene-dose-dependent brain phenotypes. Pervasive effects on cortical surface area imply that this copy number variant affects brain structure early in the course of development. SIGNIFICANCE STATEMENT Probing naturally occurring reciprocal copy number variation in the genome may help us understand mechanisms underlying deviations from typical brain and cognitive development. The 22q11.2 genomic region is particularly susceptible to chromosomal rearrangements and contains many genes crucial for neuronal development and migration. Not surprisingly, reciprocal genomic imbalances at this locus confer some of the highest known genetic risks for developmental neuropsychiatric disorders. Here we provide the first evidence that brain morphology differs meaningfully as a function of reciprocal genomic variation at the 22q11.2 locus. Cortical thickness and surface area were affected in opposite directions with more widespread effects of gene dosage on cortical surface area.

Endocytic adaptor epidermal growth factor receptor substrate 15 (Eps15) is involved in the trafficking of ubiquitinated α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

Background: AMPAR trafficking plays an important role in synaptic plasticity, but how ubiquitinated AMPARs internalize remains largely unknown. Results: The endocytic adaptor EPS15 interacts with ubiquitinated AMPARs and facilitates receptor internalization. Conclusion: EPS15 is required for the internalization of ubiquitinated AMPARs. Significance: Selective targeting of a distinct pool of surface AMPARs for internalization provides novel insights into the mechanisms of synaptic regulation. AMPA-type glutamate receptors (AMPARs) play a critical role in mediating fast excitatory synaptic transmission in the brain. Alterations in receptor expression, distribution, and trafficking have been shown to underlie synaptic plasticity and higher brain functions, including learning and memory, as well as brain dysfunctions such as drug addiction and psychological disorders. Therefore, it is essential to elucidate the molecular mechanisms that regulate AMPAR dynamics. We have shown previously that mammalian AMPARs are subject to posttranslational modification by ubiquitin, with AMPAR ubiquitination enhancing receptor internalization and reducing AMPAR cell surface expression. Here we report a crucial role for epidermal growth factor receptor substrate 15 (Eps15), an endocytic adaptor, in ubiquitination-dependent AMPAR internalization. We find that suppression or overexpression of Eps15 results in changes in AMPAR surface expression. Eps15 interacts with AMPARs, which requires Nedd4-mediated GluA1 ubiquitination and the ubiquitin-interacting motif of Eps15. Importantly, we find that Eps15 plays an important role in AMPAR internalization. Knockdown of Eps15 suppresses the internalization of GluA1 but not the mutant GluA1 that lacks ubiquitination sites, indicating a role of Eps15 for the internalization of ubiquitinated AMPARs. These results reveal a novel molecular mechanism employed specifically for the trafficking of the ubiquitin-modified AMPARs.

Alterations in expression and phosphorylation of GluA1 receptors following cocaine-cue extinction learning

Brain regional analyses of total GluA1 and GluA1-pSer(845) were used to delineate plasticity of the AMPA receptor in conjunction with cocaine-cue extinction learning. Rats were trained to self-administer cocaine paired with a 2-s light cue and later underwent a single 2 h extinction session for which cocaine was withheld but response-contingent cues were presented. Control groups received yoked-saline sessions or received cocaine self-administration training without undergoing extinction training. Extinction-related increases and decreases, respectively, in total GluA1 were observed in the ventromedial prefrontal cortex (vmPFC) and basolateral amygdala (BLA). Phosphorylation of GluA1 at Ser(845) was increased in the vmPFC and nucleus accumbens (NAc). Though total GluA1 did not change in NAc, there was a positive association between the number of responses during extinction training and the magnitude of total GluA1 in NAc. No significant changes were evident in the dorsal hippocampus. We conclude that the BLA and vmPFC, in particular, appear to be loci for the inhibition of learned behavior induced via extinction training, but each site may have different signaling functions for cocaine-cue extinction learning.