Paul L. Greer

From synapse to nucleus: calcium-dependent gene transcription in the control of synapse development and function.

One of the unique characteristics of higher organisms is their ability to learn and adapt to changes in their environment. This plasticity is largely a result of the brains ability to convert transient stimuli into long-lasting alterations in neuronal structure and function. This process is complex and involves changes in receptor trafficking, local mRNA translation, protein turnover, and new gene synthesis. Here, we review how neuronal activity triggers calcium-dependent gene expression to regulate synapse development, maturation, and refinement. Interestingly, many components of the activity-dependent gene expression program are mutated in human cognitive disorders, which suggest that this program is essential for proper brain development and function.

Activity-Dependent Regulation of MEF2 Transcription Factors Suppresses Excitatory Synapse Number

In the mammalian nervous system, neuronal activity regulates the strength and number of synapses formed. The genetic program that coordinates this process is poorly understood. We show that myocyte enhancer factor 2 (MEF2) transcription factors suppressed excitatory synapse number in a neuronal activity- and calcineurin-dependent manner as hippocampal neurons formed synapses. In response to increased neuronal activity, calcium influx into neurons induced the activation of the calcium/calmodulin-regulated phosphatase calcineurin, which dephosphorylated and activated MEF2. When activated, MEF2 promoted the transcription of a set of genes, including arc and synGAP, that restrict synapse number. These findings define an activity-dependent transcriptional program that may control synapse number during development.

The Angelman Syndrome Protein Ube3A Regulates Synapse Development by Ubiquitinating Arc

Angelman Syndrome is a debilitating neurological disorder caused by mutation of the E3 ubiquitin ligase Ube3A, a gene whose mutation has also recently been associated with autism spectrum disorders (ASDs). The function of Ube3A during nervous system development and how Ube3A mutations give rise to cognitive impairment in individuals with Angleman Syndrome and ASDs are not clear. We report here that experience-driven neuronal activity induces Ube3A transcription and that Ube3A then regulates excitatory synapse development by controlling the degradation of Arc, a synaptic protein that promotes the internalization of the AMPA subtype of glutamate receptors. We find that disruption of Ube3A function in neurons leads to an increase in Arc expression and a concomitant decrease in the number of AMPA receptors at excitatory synapses. We propose that this deregulation of AMPA receptor expression at synapses may contribute to the cognitive dysfunction that occurs in Angelman Syndrome and possibly other ASDs.

Vav Family GEFs Link Activated Ephs to Endocytosis and Axon Guidance

Ephrin signaling through Eph receptor tyrosine kinases can promote attraction or repulsion of axonal growth cones during development. However, the mechanisms that determine whether Eph signaling promotes attraction or repulsion are not known. We show here that the Rho family GEF Vav2 plays a key role in this process. We find that, during axon guidance, ephrin binding to Ephs triggers Vav-dependent endocytosis of the ligand-receptor complex, thus converting an initially adhesive interaction into a repulsive event. In the absence of Vav proteins, ephrin-Eph endocytosis is blocked, leading to defects in growth cone collapse in vitro and significant defects in the ipsilateral retinogeniculate projections in vivo. These findings suggest an important role for Vav family GEFs as regulators of ligand-receptor endocytosis and determinants of repulsive signaling during axon guidance.

Eph-dependent tyrosine phosphorylation of ephexin1 modulates growth cone collapse

Ephs regulate growth cone repulsion, a process controlled by the actin cytoskeleton. The guanine nucleotide exchange factor (GEF) ephexin1 interacts with EphA4 and has been suggested to mediate the effect of EphA on the activity of Rho GTPases, key regulators of the cytoskeleton and axon guidance. Using cultured ephexin1-/- mouse neurons and RNA interference in the chick, we report that ephexin1 is required for normal axon outgrowth and ephrin-dependent axon repulsion. Ephexin1 becomes tyrosine phosphorylated in response to EphA signaling in neurons, and this phosphorylation event is required for growth cone collapse. Tyrosine phosphorylation of ephexin1 enhances ephexin1s GEF activity toward RhoA while not altering its activity toward Rac1 or Cdc42, thus changing the balance of GTPase activities. These findings reveal that ephexin1 plays a role in axon guidance and is regulated by a switch mechanism that is specifically tailored to control Eph-mediated growth cone collapse.

EphB-mediated degradation of the RhoA GEF Ephexin5 relieves a developmental brake on excitatory synapse formation

The mechanisms that promote excitatory synapse formation and maturation have been extensively studied. However, the molecular events that limit excitatory synapse development so that synapses form at the right time and place and in the correct numbers are less well understood. We have identified a RhoA guanine nucleotide exchange factor, Ephexin5, which negatively regulates excitatory synapse development until EphrinB binding to the EphB receptor tyrosine kinase triggers Ephexin5 phosphorylation, ubiquitination, and degradation. The degradation of Ephexin5 promotes EphB-dependent excitatory synapse development and is mediated by Ube3A, a ubiquitin ligase that is mutated in the human cognitive disorder Angelman syndrome and duplicated in some forms of Autism Spectrum Disorders (ASDs). These findings suggest that aberrant EphB/Ephexin5 signaling during the development of synapses may contribute to the abnormal cognitive function that occurs in Angelman syndrome and, possibly, ASDs.

The Cyclin-Dependent Kinase 5 Activators p35 and p39 Interact with the α-Subunit of Ca2+/Calmodulin-Dependent Protein Kinase II and α-Actinin-1 in a Calcium-Dependent Manner

Cyclin-dependent kinase 5 (Cdk5) is a critical regulator of neuronal migration in the developing CNS, and recent studies have revealed a role for Cdk5 in synaptogenesis and regulation of synaptic transmission. Deregulation of Cdk5 has been linked to the pathology of neurodegenerative diseases such as Alzheimers disease. Activation of Cdk5 requires its association with a regulatory subunit, and two Cdk5 activators, p35 and p39, have been identified. To gain further insight into the functions of Cdk5, we identified proteins that interact with p39 in a yeast two-hybrid screen. In this study we report that α-actinin-1 and the α-subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKIIα), two proteins localized at the postsynaptic density, interact with Cdk5 via their association with p35 and p39. CaMKIIα and α-actinin-1 bind to distinct regions of p35 and p39 and also can interact with each other. The association of CaMKIIα and α-actinin-1 to the Cdk5 activators, as well as to each other, is stimulated by calcium. Further, the activation of glutamate receptors increases the association of p35 and p39 with CaMKIIα, and the inhibition of CaMKII activation diminishes this effect. The glutamate-mediated increase in association of p35 and CaMKIIα is mediated in large part by NMDA receptors, suggesting that cross talk between the Cdk5 and CaMKII signal transduction pathways may be a component of the complex molecular mechanisms contributing to synaptic plasticity, memory, and learning.

SnapShot: Ca2+-Dependent Transcription in Neurons

Synaptic activity stimulates the influx of calcium ions into the postsynaptic neuron and thereby sets in motion a cascade of signaling events that lead to changes in gene expression. These changes in gene expression affect many aspects of nervous system development including dendritic morphogenesis, neuronal survival, and synapse development as well as the adaptive responses that underlie learning and memory in the mature nervous system. Mutations in components of the signal-ing pathways that participate in the process of experience-dependent brain development have been found to give rise to a variety of disorders of cognitive function including autism spectrum disorders.The initial contact between the axon and the dendrite during synapse development is mediated by cell surface-associated proteins on the pre- and postsynaptic membranes. For example, the binding of presynaptic β-neurexin to its postsynaptic receptor, Neuroligin1, leads to the recruitment of PSD-95 at nascent excitatory synapses. Ephrin/Eph signaling leads to the recruitment of additional proteins and the potentiation of NMDA receptor signaling. Release of the excitatory neurotrans-mitter glutamate from the presynaptic membrane and its binding to NMDA receptors and AMPA receptors on the postsynaptic membrane lead to the opening of these glutamate-gated ion channels. This is followed by membrane depolarization, opening of the L-type voltage-gated calcium ion channel (L-VSCC), and a rapid rise in calcium ions in the postsynaptic neuron as well as other local changes including protein recruitment and activation. Calcium entry through L-VSCCs leads to the recruitment of AKAP79/150, which then recruits PKA to the channel. PKA phosphorylates the calcium channel, thereby increasing its ability to allow calcium ions to flow into the cell. Calcium ion influx through L-VSCCs is sensed by calmodulin (CaM). Activated calmodulin initiates a cascade of events including stimulation of the guanine nucleotide exchange factor RasGRF, followed by activation of the Ras-MAPK signaling cascade. Calcium-activated calmodulin also activates the CaM kinase signaling pathway. Once activated these pathways trigger the phosphorylation and activation of a wide range of transcription factors such as CREB and NeuroD2. The phosphorylation of these transcription factors occurs in the nucleus and can be triggered by a cascade of events that begins at the site of calcium entry at the mouth of the calcium channel (that is, the Ras, Raf, MEK, ERK, RSK/MSK signaling pathway). Alternatively, channel activation can trigger an elevation of calcium ions directly in the nucleus that leads to activation of nuclear CaMKII by calcium/calmodulin, which in turn phosphorylates CREB and NeuroD2. In addition, dephosphorylation-dependent signaling through calcium/calmodulin activation of calcineurin leads to the activation of the transcripton factors NFAT and MEF2.Once activated and localized to the nucleus, calcium-activated transcription factors and modulators of transcription bind to the regulatory regions of activity-regulated genes to orchestrate finely tuned levels of gene expression. The most extensively studied activity-regulated gene is

Shh-proteoglycan interactions regulate maturation of olfactory glomerular circuitry

The olfactory system relies on precise circuitry connecting olfactory sensory neurons (OSNs) and appropriate relay and processing neurons of the olfactory bulb (OB). In mammals, the exact correspondence between specific olfactory receptor types and individual glomeruli enables a spatially precise map of glomerular activation that corresponds to distinct odors. However, the mechanisms that govern the establishment and maintenance of the glomerular circuitry are largely unknown. Here we show that high levels of Sonic Hedgehog (Shh) signaling at multiple sites enable refinement and maintenance of olfactory glomerular circuitry. Mice expressing a mutant version of Shh (ShhAla/Ala), with impaired binding to proteoglycan co‐receptors, exhibit disproportionately small olfactory bulbs containing fewer glomeruli. Notably, in mutant animals the correspondence between individual glomeruli and specific olfactory receptors is lost, as olfactory sensory neurons expressing different olfactory receptors converge on the same glomeruli. These deficits arise at late stages in post‐natal development and continue into adulthood, indicating impaired pruning of erroneous connections within the olfactory bulb. In addition, mature ShhAla/Ala mice exhibit decreased proliferation in the subventricular zone (SVZ), with particular reduction in neurogenesis of calbindin‐expressing periglomerular cells. Thus, Shh interactions with proteoglycan co‐receptors function at multiple locations to regulate neurogenesis and precise olfactory connectivity, thereby promoting functional neuronal circuitry.