David G. Wells

CPEB-Mediated Cytoplasmic Polyadenylation and the Regulation of Experience-Dependent Translation of α-CaMKII mRNA at Synapses

Long-term changes in synaptic efficacy may require the regulated translation of dendritic mRNAs. While the basis of such regulation is unknown, it seemed possible that some features of translational control in development could be recapitulated in neurons. Polyadenylation-induced translation of oocyte mRNAs requires the cis-acting CPE sequence and the CPE-binding protein CPEB. CPEB is also present in the dendritic layers of the hippocampus, at synapses in cultured neurons, and in postsynaptic densities of adult brain. alpha-CaMKII mRNA, which is localized in dendrites and is necessary for synaptic plasticity and LTP, contains two CPEs. These CPEs are bound by CPEB and mediate polyadenylation-induced translation in injected Xenopus oocytes. In the intact brain, visual experience induces alpha-CaMKII mRNA polyadenylation and translation, suggesting that this process likely occurs at synapses.

SAP90 Binds and Clusters Kainate Receptors Causing Incomplete Desensitization

The mechanism of kainate receptor targeting and clustering is still unresolved. Here, we demonstrate that members of the SAP90/PSD-95 family colocalize and associate with kainate receptors. SAP90 and SAP102 coimmunoprecipitate with both KA2 and GluR6, but only SAP97 coimmunoprecipitates with GluR6. Similar to NMDA receptors, GluR6 clustering is mediated by the interaction of its C-terminal amino acid sequence, ETMA, with the PDZ1 domain of SAP90. In contrast, the KA2 C-terminal region binds to, and is clustered by, the SH3 and GK domains of SAP90. Finally, we show that SAP90 coexpressed with GluR6 or GluR6/KA2 receptors alters receptor function by reducing desensitization. These studies suggest that the organization and electrophysiological properties of synaptic kainate receptors are modified by association with members of the SAP90/PSD-95 family.

Rapid, Activity-Induced Increase in Tissue Plasminogen Activator Is Mediated by Metabotropic Glutamate Receptor-Dependent mRNA Translation

Long-term synaptic plasticity is both protein synthesis-dependent and synapse-specific. Therefore, the identity of the newly synthesized proteins, their localization, and mechanism of regulation are critical to our understanding of this process. Tissue plasminogen activator (tPA) is a secreted protease required for some forms of long-term synaptic plasticity. Here, we show tPA activity is rapidly increased in hippocampal neurons after glutamate stimulation. This increase in tPA activity corresponds to an increase in tPA protein synthesis that results from the translational activation of mRNA present at the time of stimulation. Furthermore, the mRNA encoding tPA is present in dendrites and is rapidly polyadenylated after glutamate stimulation. Both the polyadenylation of tPA mRNA and the subsequent increase in tPA protein is dependent on metabotropic glutamate receptor (mGluR) activation. A similar mGluR-dependent increase in tPA activity was detected after stimulation of a synaptic fraction isolated from the hippocampus, suggesting tPA synthesis is occurring in the synaptodendritic region. Finally, we demonstrate that tPA mRNA is bound by the mRNA-binding protein CPEB (cytoplasmic polyadenylation element binding protein-1), a protein known to regulate mRNA translation via polyadenylation. These results indicate that neurons are capable of synthesizing a secreted protein in the synaptic region, that mGluR activation induces mRNA polyadenylation and translation of specific mRNA, and suggest a model for synaptic plasticity whereby translational regulation of an immediate early gene precedes the increase in gene transcription.

Molecular mechanisms for activity-regulated protein synthesis in the synapto-dendritic compartment.

The creation of enduring modifications in synaptic efficacy requires new protein synthesis. Neurons face the formidable challenge of directing these newly made proteins to the appropriate subset of synapses. One attractive solution to this problem is the local translation of mRNAs that are targeted to dendrites and perhaps to synapses themselves. The molecular mechanisms mediating such local protein synthesis, notably CPEB-mediated cytoplasmic polyadenylation, are now being elucidated.

Clonally expanded CD4+ T cells can produce infectious HIV-1 in vivo

Significance Reservoirs of HIV-infected cells persist during antiretroviral therapy, and understanding persistence is essential to develop HIV curative strategies. During replication, HIV integrates into the host genome; most proviruses are not infectious, but some with replication-competent HIV persist. Cells with integrated HIV can proliferate, potentially expanding the reservoir, but whether cells with replication-competent HIV actually undergo expansion is unknown. HIV reactivation is often lethal to infected cells, and others have reported finding no replication-competent HIV in expanded populations. We describe a highly expanded clone containing infectious HIV that was the source of viremia for years in a patient. Clonally expanded populations can represent a long-lived reservoir of HIV. Curative strategies will require targeting this persistence mechanism. Reservoirs of infectious HIV-1 persist despite years of combination antiretroviral therapy and make curing HIV-1 infections a major challenge. Most of the proviral DNA resides in CD4+T cells. Some of these CD4+T cells are clonally expanded; most of the proviruses are defective. It is not known if any of the clonally expanded cells carry replication-competent proviruses. We report that a highly expanded CD4+ T-cell clone contains an intact provirus. The highly expanded clone produced infectious virus that was detected as persistent plasma viremia during cART in an HIV-1–infected patient who had squamous cell cancer. Cells containing the intact provirus were widely distributed and significantly enriched in cancer metastases. These results show that clonally expanded CD4+T cells can be a reservoir of infectious HIV-1.

Cytoplasmic polyadenylation element binding protein regulates neurotrophin 3-dependent β-catenin mRNA translation in developing hippocampal neurons

Neuronal morphogenesis, the growth and arborization of neuronal processes, is an essential component of brain development. Two important but seemingly disparate components regulating neuronal morphology have previously been described. In the hippocampus, neurotrophins, particularly brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3), act to enhance cell growth and branching, while activity-induced branching was shown to be dependent upon intracellular β-catenin. We now describe a molecular link between NT3 stimulation and β-catenin increase in developing neurons and demonstrate that this process is required for the NT3-mediated increase in process branching. Here, we show that β-catenin is rapidly increased specifically in growth cones following NT3 stimulation. This increase in β-catenin is protein synthesis dependent and requires the activity of cytoplasmic polyadenylation element-binding protein-1 (CPEB1), an mRNA-binding protein that regulates mRNA translation. We find that CPEB1 protein binds β-catenin mRNA in a CPE-dependent manner and that both localize to growth cones of developing hippocampal neurons. Both the NT3-mediated rapid increase in β-catenin and process branching are abolished when CPEB1 function is inhibited. In addition, the NT3-mediated increase in β-catenin in growth cones is dependent upon internal calcium and the activity of CaMKII (calcium/calmodulin-dependent kinase II). Together, these results suggest that CPEB1 regulates β-catenin synthesis in neurons and may contribute to neuronal morphogenesis.

Cytoplasmic Polyadenylation Element Binding Protein 1-Mediated mRNA Translation in Purkinje Neurons Is Required for Cerebellar Long-Term Depression and Motor Coordination

The ability of neurons to modify synaptic connections is critical for proper brain development and function in the adult. It is now clear that changes in synaptic strength are often accompanied by changes in synaptic morphology. This synaptic plasticity can be maintained for varying lengths of time depending on the type of neuronal activity that first induced the changes. Long-term synaptic plasticity requires the synthesis of new proteins, and one mechanism for the regulation of experience-induced protein synthesis in neurons involves cytoplasmic polyadenylation element binding protein (CPEB1). CPEB1 can bidirectionally regulate mRNA translation, first repressing translation, and then activating translation after the phosphorylation of two critical residues (T171 and S177). To determine the full extent of CPEB1-mediated protein synthesis in synaptic function, we engineered a line of mice expressing CPEB1 with these phosphorylation sites mutated to alanines (mCPEB1-AA) exclusively in cerebellar Purkinje neurons (PNs). Thus, mRNAs bound by mCPEB1-AA would be held in a translationally dormant state. We show that mCPEB1-AA localizes to synapses in cerebellum and resulted in a loss of protein synthesis-dependent phase of parallel fiber–PN long-term depression. This was accompanied by a change in spine number and spine length that are likely attributable in part to the dysregulation of IRSp53, a protein known to play a role in synaptic structure. Finally, mCPEB1-AA mice displayed a significant impairment of motor coordination and a motor learning delay.

Dendritic mRNA translation: deciphering the uncoded

The interaction of two untranslated sequences in the mRNA for calcium/calmodulin-dependent protein kinase II could regulate its activity-dependent transport into dendrites.

In search of molecular memory: experience-driven protein synthesis.

The formation and maintenance of memories is one of the most intriguing functions of the brain. The human brain has the remarkable ability to store and retrieve past experiences for years and in some instances decades. Of late, extensive effort has been put into reducing the process of learning and memory to the cellular and molecular level [see ref. 1]. The underlying logic is that if we can understand how an individual neuron remembers we might then extrapolate this function to groups of neurons residing in areas of the brain involved in memory formation and storage. One such area, residing in the medial temporal lobe, is called the hippocampus. This structure is especially important for explicit or declarative memory—the recall of information pertaining to people or places [2]. The process of protein synthesis is a critical event in the encoding of long-term (explicit) memory. Thus, a great deal of research emphasis has been placed on the molecular basis of this experience-driven protein synthesis in the hope that it will elucidate the foundations of memory. The principal loci of cell-cell communication in the brain are synapses. Since synaptic transmission encodes information in the brain, the engram for memory may lie in the ability of the synapse to use stable modifications to remember its excitatory history. This form of synaptic plasticity was postulated in 1949 by Hebb [3], and was first described experimentally in the early 1970s as a long-lasting increase in synaptic strength following robust synaptic activity [4, 5]. The long-lasting potentiation (now called long-term potentiation or LTP) that Bliss and colleagues described in the hippocampus has become integral to our present theories for how memories are encoded [6, 7]. Although much of the following discussion centers on LTP, it is important to note that synaptic plasticity is a bidirectional process and that a synaptic weakening or long-term depression (LTD) is likely to play a role in memory as well [8, 9].

Cyclin B1 expression regulated by cytoplasmic polyadenylation element binding protein in astrocytes

Astrocytes are the most abundant cells in the brain, playing vital roles in neuronal survival, growth, and function. Understanding the mechanism(s) regulating astrocyte proliferation will have important implications in brain development, response to injury, and tumorigenesis. Cyclin B1 is well known to be a critical regulator of mitotic entry via its interaction with cyclin-dependent kinase 1. In rat astrocytes, we now show that the mRNA binding protein cytoplasmic polyadenylation element binding protein 1 (CPEB1) is associated with cyclin B1 mRNA and that this interaction is enriched at the centrosome. In addition, if growth-arrested astrocytes are stimulated to divide, CPEB1 is phosphorylated and cyclin B1 mRNA is polyadenylated, both hallmarks of CPEB1 activation, resulting in an increase in cyclin B1 protein. CPEB1 binding to mRNA initially inhibits translation; therefore, removing CPEB1 from mRNA should result in an increase in translation due to derepression. Indeed, when we either knocked down CPEB1 protein with siRNA or sequestered it from endogenous mRNA by expressing RNA containing multiple CPEB1 binding sites, cyclin B1 protein was increased and cell proliferation was stimulated. Our data suggest a mechanism wherein CPEB1 is bound and represses cyclin B1 mRNA translation until a signal to proliferate phosphorylates CPEB1, resulting in an increase in cyclin B1 protein and progression into mitosis. Our results demonstrate for the first time a role for CPEB1 in regulating cell proliferation in the brain.