Copper is an endogenous modulator of neural circuit spontaneous activity.

For reasons that remain insufficiently understood, the brain requires among the highest levels of metals in the body for normal function. The traditional paradigm for this organ and others is that fluxes of alkali and alkaline earth metals are required for signaling, but transition metals are maintained in static, tightly bound reservoirs for metabolism and protection against oxidative stress. Here we show that copper is an endogenous modulator of spontaneous activity, a property of functional neural circuitry. Using Copper Fluor-3 (CF3), a new fluorescent Cu(+) sensor for one- and two-photon imaging, we show that neurons and neural tissue maintain basal stores of loosely bound copper that can be attenuated by chelation, which define a labile copper pool. Targeted disruption of these labile copper stores by acute chelation or genetic knockdown of the CTR1 (copper transporter 1) copper channel alters the spatiotemporal properties of spontaneous activity in developing hippocampal and retinal circuits. The data identify an essential role for copper neuronal function and suggest broader contributions of this transition metal to cell signaling.

Mechanism and treatment for learning and memory deficits in mouse models of Noonan syndrome.

In Noonan syndrome (NS) 30-50% of subjects show cognitive deficits of unknown etiology and with no known treatment. Here, we report that knock-in mice expressing either of two NS-associated mutations in Ptpn11, which encodes the nonreceptor protein tyrosine phosphatase Shp2, show hippocampal-dependent impairments in spatial learning and deficits in hippocampal long-term potentiation (LTP). In addition, viral overexpression of an NS-associated allele PTPN11(D61G) in adult mouse hippocampus results in increased baseline excitatory synaptic function and deficits in LTP and spatial learning, which can be reversed by a mitogen-activated protein kinase kinase (MEK) inhibitor. Furthermore, brief treatment with lovastatin reduces activation of the GTPase Ras-extracellular signal-related kinase (Erk) pathway in the brain and normalizes deficits in LTP and learning in adult Ptpn11(D61G/+) mice. Our results demonstrate that increased basal Erk activity and corresponding baseline increases in excitatory synaptic function are responsible for the LTP impairments and, consequently, the learning deficits in mouse models of NS. These data also suggest that lovastatin or MEK inhibitors may be useful for treating the cognitive deficits in NS.

Calcium-dependent copper redistributions in neuronal cells revealed by a fluorescent copper sensor and X-ray fluorescence microscopy.

Dynamic fluxes of s-block metals like potassium, sodium, and calcium are of broad importance in cell signaling. In contrast, the concept of mobile transition metals triggered by cell activation remains insufficiently explored, in large part because metals like copper and iron are typically studied as static cellular nutrients and there are a lack of direct, selective methods for monitoring their distributions in living cells. To help meet this need, we now report Coppersensor-3 (CS3), a bright small-molecule fluorescent probe that offers the unique capability to image labile copper pools in living cells at endogenous, basal levels. We use this chemical tool in conjunction with synchotron-based microprobe X-ray fluorescence microscopy (XRFM) to discover that neuronal cells move significant pools of copper from their cell bodies to peripheral processes upon their activation. Moreover, further CS3 and XRFM imaging experiments show that these dynamic copper redistributions are dependent on calcium release, establishing a link between mobile copper and major cell signaling pathways. By providing a small-molecule fluorophore that is selective and sensitive enough to image labile copper pools in living cells under basal conditions, CS3 opens opportunities for discovering and elucidating functions of copper in living systems.

Organelle-targetable fluorescent probes for imaging hydrogen peroxide in living cells via SNAP-Tag protein labeling.

Hydrogen peroxide (H(2)O(2)) is a potent small-molecule oxidant that can exert a diverse array of physiological and/or pathological effects within living systems depending on the timing and location of its production, accumulation, trafficking, and consumption. To help study the chemistry and biology of this reactive oxygen species (ROS) in its native cellular context, we now present a new method for monitoring local, subcellular changes in H(2)O(2) levels by fluorescence imaging. Specifically, we have exploited the versatility of the SNAP-tag technology for site-specific protein labeling with small molecules on the surface or interior of living cells with the use of boronate-capped dyes to selectively visualize H(2)O(2). The resulting SNAP-Peroxy-Green (SNAP-PG) probes consist of appropriately derivatized boronates bioconjugated to SNAP-tag fusion proteins. Spectroscopic measurements of the SNAP-PG constructs confirm their ability to detect H(2)O(2) with specificity over other biologically relevant ROS. Moreover, these hybrid small-molecule/protein reporters can be used in live mammalian cells expressing SNAP-tag fusion proteins directed to the plasma membrane, nucleus, mitochondria, and endoplasmic reticulum. Imaging experiments using scanning confocal microscopy establish organelle-specific localization of the SNAP-tag probes and their fluorescence turn-on in response to changes in local H(2)O(2) levels. This work provides a general molecular imaging platform for assaying H(2)O(2) chemistry in living cells with subcellular resolution.

Synaptic signaling by all-trans retinoic acid in homeostatic synaptic plasticity.

Normal brain function requires that the overall synaptic activity in neural circuits be kept constant. Long-term alterations of neural activity lead to homeostatic regulation of synaptic strength by a process known as synaptic scaling. The molecular mechanisms underlying synaptic scaling are largely unknown. Here, we report that all-trans retinoic acid (RA), a well-known developmental morphogen, unexpectedly mediates synaptic scaling in response to activity blockade. We show that activity blockade increases RA synthesis in neurons and that acute RA treatment enhances synaptic transmission. The RA-induced increase in synaptic strength is occluded by activity blockade-induced synaptic scaling. Suppression of RA synthesis prevents synaptic scaling. This form of RA signaling operates via a translation-dependent but transcription-independent mechanism, causes an upregulation of postsynaptic glutamate receptor levels, and requires RARalpha receptors. Together, our data suggest that RA functions in homeostatic plasticity as a signaling molecule that increases synaptic strength by a protein synthesis-dependent mechanism.

Retinoic acid regulates RARalpha-mediated control of translation in dendritic RNA granules during homeostatic synaptic plasticity.

Homeostatic plasticity is thought to play an important role in maintaining the stability of neuronal circuits. During one form of homeostatic plasticity, referred to as synaptic scaling, activity blockade leads to a compensatory increase in synaptic transmission by stimulating in dendrites the local translation and synaptic insertion of the AMPA receptor subunit GluR1. We have previously shown that all-trans retinoic acid (RA) mediates activity blockade-induced synaptic scaling by activating dendritic GluR1 synthesis and that this process requires RARalpha, a member of the nuclear RA receptor family. This result raised the question of where RARalpha is localized in dendrites and whether its localization is regulated by RA and/or activity blockade. Here, we show that activity blockade or RA treatment in neurons enhances the concentration of RARalpha in the dendritic RNA granules and activates local GluR1 synthesis in these RNA granules. Importantly, the same RNA granules that contain RARalpha also exhibit an accumulation of GluR1 protein but with a much slower time course than that of RARalpha, suggesting that the former regulates the latter. Taken together, our results provide a direct link between dendritically localized RARalpha and local GluR1 synthesis in RNA granules during RA-mediated synaptic signaling in homeostatic synaptic plasticity.

Dynamics of postsynaptic glutamate receptor targeting.

Targeting of glutamate receptors to synapses is an important event in both developing and mature neurons. Glutamate receptors are delivered to nascent synapses during synaptogenesis and to existing synapses during activity-dependent synaptic strengthening. Increasing evidence suggests that glutamate receptors are inserted into the plasma membrane before they accumulate at the synapse. Lateral diffusion of receptors occurs at both synaptic and non-synaptic membranes, and glutamate receptors can exchange rapidly between synaptic and extrasynaptic sites. In addition, recent studies show that postsynaptic scaffold molecules can be highly mobile. The dynamic nature of the synapse suggests that many mechanisms might be involved in regulating synapse formation and synaptic plasticity.

Postsynaptic assembly induced by neurexin-neuroligin interaction and neurotransmitter.

Presynaptic and postsynaptic differentiation occurs at axodendritic contacts between CNS neurons. Synaptic adhesion mediated by synaptic cell adhesion molecule (SynCAM) and beta-neurexins/neuroligins triggers presynaptic differentiation. The signals that trigger postsynaptic differentiation are, however, unknown. Here we report that beta-neurexin expressed in nonneuronal cells induced postsynaptic density (PSD)-95 clustering in contacting dendrites of hippocampal neurons. The effect is specific to beta-neurexin and was not observed with other synaptic cell adhesion molecules such as N-cadherin or SynCAM. NMDA receptors, but not alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptors (AMPARs), were recruited to this beta-neurexin-induced PSD-95 scaffold. Remarkably, AMPARs were inserted into this scaffold upon glutamate application or expression of a constitutively active form of calmodulin kinase II in neurons. Expression of a dominant-negative neuroligin-1 in cultured neurons markedly reduced the sizes and densities of PSD-95 puncta and AMPAR clusters. In addition, excitatory, but not inhibitory, synaptic functions were impaired in these neurons, confirming that PSD-95/neuroligin-1 interaction is involved in postsynaptic assembly at glutamatergic synapses. These results demonstrate that postsynaptic assembly of the glutamatergic synapse may be initiated by presynaptic beta-neurexin and that glutamate release also is required for maturation of synapses.