Valentina Mercaldo

A new function for the fragile X mental retardation protein in regulation of PSD-95 mRNA stability

Fragile X syndrome (FXS) results from the loss of the fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates a variety of cytoplasmic mRNAs. FMRP regulates mRNA translation and may be important in mRNA localization to dendrites. We report a third cytoplasmic regulatory function for FMRP: control of mRNA stability. In mice, we found that FMRP binds, in vivo, the mRNA encoding PSD-95, a key molecule that regulates neuronal synaptic signaling and learning. This interaction occurs through the 3′ untranslated region of the PSD-95 (also known as Dlg4) mRNA, increasing message stability. Moreover, stabilization is further increased by mGluR activation. Although we also found that the PSD-95 mRNA is synaptically localized in vivo, localization occurs independently of FMRP. Through our functional analysis of this FMRP target we provide evidence that dysregulation of mRNA stability may contribute to the cognitive impairments in individuals with FXS.

Upregulation of CREB-Mediated Transcription Enhances Both Short- and Long-Term Memory

Unraveling the mechanisms by which the molecular manipulation of genes of interest enhances cognitive function is important to establish genetic therapies for cognitive disorders. Although CREB is thought to positively regulate formation of long-term memory (LTM), gain-of-function effects of CREB remain poorly understood, especially at the behavioral level. To address this, we generated four lines of transgenic mice expressing dominant active CREB mutants (CREB-Y134F or CREB-DIEDML) in the forebrain that exhibited moderate upregulation of CREB activity. These transgenic lines improved not only LTM but also long-lasting long-term potentiation in the CA1 area in the hippocampus. However, we also observed enhanced short-term memory (STM) in contextual fear-conditioning and social recognition tasks. Enhanced LTM and STM could be dissociated behaviorally in these four lines of transgenic mice, suggesting that the underlying mechanism for enhanced STM and LTM are distinct. LTM enhancement seems to be attributable to the improvement of memory consolidation by the upregulation of CREB transcriptional activity, whereas higher basal levels of BDNF, a CREB target gene, predicted enhanced shorter-term memory. The importance of BDNF in STM was verified by microinfusing BDNF or BDNF inhibitors into the hippocampus of wild-type or transgenic mice. Additionally, increasing BDNF further enhanced LTM in one of the lines of transgenic mice that displayed a normal BDNF level but enhanced LTM, suggesting that upregulation of BDNF and CREB activity cooperatively enhances LTM formation. Our findings suggest that CREB positively regulates memory consolidation and affects memory performance by regulating BDNF expression.

Neurons Are Recruited to a Memory Trace Based on Relative Neuronal Excitability Immediately before Training

Memories are thought to be sparsely encoded in neuronal networks, but little is known about why a given neuron is recruited or allocated to a particular memory trace. Previous research shows that in the lateral amygdala (LA), neurons with increased CREB are selectively recruited to a fear memory trace. CREB is a ubiquitous transcription factor implicated in many cellular processes. Which process mediates neuronal memory allocation? One hypothesis is that CREB increases neuronal excitability to bias neuronal recruitment, although this has not been shown experimentally. Here we use several methods to increase neuronal excitability and show this both biases recruitment into the memory trace and enhances memory formation. Moreover, artificial activation of these neurons alone is a sufficient retrieval cue for fear memory expression, showing that these neurons are critical components of the memory trace. These results indicate that neuronal memory allocation is based on relative neuronal excitability immediately before training.

Abnormal Striatal GABA Transmission in the Mouse Model for the Fragile X Syndrome

BACKGROUND Structural and functional neuroimaging studies suggest abnormal activity in the striatum of patients with the fragile X syndrome (FXS), the most common form of inherited mental retardation. METHODS Neurophysiological and immunofluorescence experiments in striatal brain slices. We studied the synaptic transmission in a mouse model for FXS, as well as the subcellular localization of fragile X mental retardation protein (FMRP) and brain cytoplasmic (BC1) RNA in striatal axons. RESULTS Our results show that absence of FMRP is associated with apparently normal striatal glutamate-mediated transmission, but abnormal gamma-aminobutyric acid (GABA) transmission. This effect is likely secondary to increased transmitter release from GABAergic nerve terminals. We detected the presence of FMRP in axons of striatal neurons and observed a selective increase in the frequency of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs, mIPSCs) in fmr1-knockout mice. We also observed reduced paired-pulse ratio of evoked IPSCs, a finding that is consistent with the idea that transmitter release probability from striatal GABAergic nerve terminals is higher than normal in these mutants. Finally, we have identified the small noncoding BC1 RNA as a critical coplayer of FMRP in the regulation of striatal synaptic transmission. CONCLUSIONS Understanding the physiologic action of FMRP and the synaptic defects associated with GABA transmission might be useful to design appropriate pharmacologic interventions for FXS.

The fragile X mental retardation protein-RNP granules show an mGluR-dependent localization in the post-synaptic spines

The localization of RNA/mRNA in dendrites plays a role in both local and temporal regulation of protein synthesis, which is required for certain forms of synaptic plasticity. A key molecule in these processes is the fragile X mental retardation protein (FMRP). Using in situ hybridization coupled to immunofluorescence confocal microscopy, we find that the FMRP-RNP particle contains alphaCaMKII and BC1 RNAs as well as Staufen and CPEB proteins. Furthermore, following mGluR activation, the FMRP-mRNP complex moves into spines as shown by co-localization with the PSD-95 and Shank proteins. This study shows, for the first time, that the translationally inactive FMRP-mRNP complex relocates into neuronal spines after stimulation and that de novo protein synthesis mainly contributes to the pool of FMRP at synapses.

P2X2R purinergic receptor subunit mRNA and protein are expressed by all hypothalamic hypocretin/orexin neurons

Neurophysiologic data suggest that orexin neurons are directly excited by ATP through purinergic receptors (P2XR). Anatomical studies, though reporting P2XR in the hypothalamus, did not describe it in the perifornical hypothalamic area, where orexinergic neurons are located. Here we report the presence of the P2X2R subunit in the rat perifornical hypothalamus and demonstrate that hypothalamic orexin neurons express the P2X2R. Double immunohistochemistry showed that virtually all orexin‐immunoreactive neurons are also P2X2R immunoreactive, whereas 80% of P2X2R‐immunoreactive neurons are also orexin positive. Triple‐labeling experiments, combining fluorescence in situ hybridization for P2X2R mRNA and P2X2R/orexin double immunofluorescence, confirmed these findings. In addition, in situ hybridization demonstrated that P2X2R mRNA is localized in cellular processes of orexinergic neurons. The present data support neurophysiologic findings on ATP modulation of orexinergic function and provide direct evidence that the entire population of orexin neurons expresses a P2XR subtype, namely, P2X2R. Thus, purinergic transmission might intervene in modulating key functions known to be controlled by the orexinergic system, such as feeding behavior and arousal. J. Comp. Neurol. 498:58–67, 2006.

Competition between engrams influences fear memory formation and recall

How to link and separate memories Engrams are the changes in brain tissue that store single memories. Neuroscientists can localize and manipulate them, but until now, little was known about how multiple engrams interact to influence memories. Rashid et al. examined how neural assemblies in an area called the lateral amygdala interact. If two frightening events occurred within 6 hours, the same set of neurons was used to express fear memories for both events. However, if the events were separated by 24 hours, distinct memory traces were formed. Science, this issue p. 383 Memories are linked or separated via a transient winner-takes-all competition for recruitment to a memory trace. Collections of cells called engrams are thought to represent memories. Although there has been progress in identifying and manipulating single engrams, little is known about how multiple engrams interact to influence memory. In lateral amygdala (LA), neurons with increased excitability during training outcompete their neighbors for allocation to an engram. We examined whether competition based on neuronal excitability also governs the interaction between engrams. Mice received two distinct fear conditioning events separated by different intervals. LA neuron excitability was optogenetically manipulated and revealed a transient competitive process that integrates memories for events occurring closely in time (coallocating overlapping populations of neurons to both engrams) and separates memories for events occurring at distal times (disallocating nonoverlapping populations to each engram).

MEF2 negatively regulates learning-induced structural plasticity and memory formation

Memory formation is thought to be mediated by dendritic-spine growth and restructuring. Myocyte enhancer factor 2 (MEF2) restricts spine growth in vitro, suggesting that this transcription factor negatively regulates the spine remodeling necessary for memory formation. Here we show that memory formation in adult mice was associated with changes in endogenous MEF2 levels and function. Locally and acutely increasing MEF2 function in the dentate gyrus blocked both learning-induced increases in spine density and spatial-memory formation. Increasing MEF2 function in amygdala disrupted fear-memory formation. We rescued MEF2-induced memory disruption by interfering with AMPA receptor endocytosis, suggesting that AMPA receptor trafficking is a key mechanism underlying the effects of MEF2. In contrast, decreasing MEF2 function in dentate gyrus and amygdala facilitated the formation of spatial and fear memory, respectively. These bidirectional effects indicate that MEF2 is a key regulator of plasticity and that relieving the suppressive effects of MEF2-mediated transcription permits memory formation.

FRAGILE X MENTAL RETARDATION PROTEIN IS REQUIRED FOR CHEMICALLY-INDUCED LONG-TERM POTENTIATION OF THE HIPPOCAMPUS IN ADULT MICE

Fragile X syndrome (FXS), a common form of inherited mental retardation, is caused by the lack of fragile X mental retardation protein (FMRP). The animal model of FXS, Fmr1 knockout mice, have deficits in the Morris water maze and trace fear memory tests, showing impairment in hippocampus‐dependent learning and memory. However, results for synaptic long‐term potentiation (LTP), a key cellular model for learning and memory, remain inconclusive in the hippocampus of Fmr1 knockout mice. Here, we demonstrate that FMRP is required for glycine induced LTP (Gly‐LTP) in the CA1 of hippocampus. This form of LTP requires activation of post‐synaptic NMDA receptors and metabotropic glutamateric receptors, as well as the subsequent activation of extracellular signal‐regulated kinase (ERK) 1/2. However, paired‐pulse facilitation was not affected by glycine treatment. Genetic deletion of FMRP interrupted the phosphorylation of ERK1/2, suggesting the possible role of FMRP in the regulation of the activity of ERK1/2. Our study provide strong evidences that FMRP participates in Gly‐LTP in the hippocampus by regulating the phosphorylation of ERK1/2, and that improper regulation of these signaling pathways may contribute to the learning and memory deficits observed in FXS.

Dendritic LSm1/CBP80-mRNPs mark the early steps of transport commitment and translational control

Messenger RNA (mRNA) transport to neuronal dendrites is crucial for synaptic plasticity, but little is known of assembly or translational regulation of dendritic messenger ribonucleoproteins (mRNPs). Here we characterize a novel mRNP complex that is found in neuronal dendrites throughout the central nervous system and in some axonal processes of the spinal cord. The complex is characterized by the LSm1 protein, which so far has been implicated in mRNA degradation in nonneuronal cells. In brain, it associates with intact mRNAs. Interestingly, the LSm1-mRNPs contain the cap-binding protein CBP80 that associates with (pre)mRNAs in the nucleus, suggesting that the dendritic LSm1 complex has been assembled in the nucleus. In support of this notion, neuronal LSm1 is partially nuclear and inhibition of mRNA synthesis increases its nuclear localization. Importantly, CBP80 is also present in the dendrites and both LSm1 and CBP80 shift significantly into the spines upon stimulation of glutamergic receptors, suggesting that these mRNPs are translationally activated and contribute to the regulated local protein synthesis.