Justin R. Fallon

Internalization of ionotropic glutamate receptors in response to mGluR activation

Activation of group 1 metabotropic glutamate receptors (mGluRs) stimulates dendritic protein synthesis and long-term synaptic depression (LTD), but it remains unclear how these effects are related. Here we provide evidence that a consequence of mGluR activation in the hippocampus is the rapid loss of both AMPA and NMDA receptors from synapses. Like mGluR-LTD, the stable expression of this change requires protein synthesis. These data suggest that expression of mGluR-LTD is at least partly postsynaptic, and that a functional consequence of dendritic protein synthesis is the regulation of glutamate receptor trafficking.

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.

Identification and purification of an agrin receptor from torpedo postsynaptic membranes: A heteromeric complex related to the dystroglycans

The selective concentration of neurotransmitter receptors at the postsynaptic membrane is an essential aspect of synaptic differentiation and function. Agrin is an extracellular matrix protein that is likely to direct the accumulation of acetylcholine receptors and several other postsynaptic elements at developing and regenerating neuromuscular junctions. How agrin interacts with the membrane to bring about these changes is unknown. We now report the identification and purification of a protein complex from Torpedo electric organ postsynaptic membranes that is likely to serve as an agrin receptor. The native receptor is a heteromeric complex of two membrane glycoproteins of 190 kDa and 50 kDa. The 190 kDa subunit is sufficient to bind ligand. Peptide sequence analysis revealed that the 190 kDa and 50 kDa subunits are related to the dystrophin-associated glycoproteins alpha- and beta-dystroglycan, respectively. No other candidate agrin receptors were detected. The identification of the agrin receptor opens new avenues toward a mechanistic understanding of synapse differentiation.

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.

Agrin and microvascular damage in Alzheimer’s disease

Abstract Heparan sulfate proteoglycans (HSPGs) are ubiquitously present within the perivascular basement membrane, and have been shown to be altered in patients with Alzheimer’s Disease (AD). Although the HSPG agrin clearly orchestrates the differentiation of the neuromuscular junction, its role in the brain remains unclear. Growing evidence suggests that agrin may be an important vascular basement membrane (VBM)-associated HSPG. In previous studies, we demonstrated that agrin is present throughout the brain microvasculature, as well as in neuronal cell bodies. AD brains exhibited fragmentation of VBM-associated agrin. Agrin immunoreactivity was also seen within senile plaques and neurofibrillary tangles. These changes were accompanied by the appearance of an additional pool of insoluble agrin. In the present study, we provide further evidence for microvascular damage in AD, by examining the distribution of agrin and laminin within the VBM, and by measuring the agrin concentration within hippocampus and prefrontal cortex. Furthermore, we assessed blood-brain-barrier (BBB) leakage by examining the perivascular distribution of prothrombin immunoreactivity. Soluble agrin levels were increased approximately 30% in Braak stage III–VI AD patients relative to age-matched controls. Furthermore, agrin and laminin exhibited identical patterns of VBM fragmentation in AD and colocalized with beta-amyloid in senile plaques. Microvascular changes were associated with the appearance of perivascular prothrombin immunoreactivity. Our data suggest that agrin is an important VBM-associated HSPG in the brain and that agrin levels are altered in association with microvascular damage in AD.

The FXG: A presynaptic Fragile X granule expressed in a subset of developing brain circuits

The loss of Fragile X mental retardation protein (FMRP) causes Fragile X syndrome, the most common inherited mental retardation and single gene cause of autism. Although postsynaptic functions for FMRP are well established, potential roles at the presynaptic apparatus remain largely unexplored. Here, we characterize the expression of FMRP and its homologs, FXR1P and FXR2P, in the developing, mature and regenerating rodent nervous system, with a focus on presynaptic expression. As expected, FMRP is expressed in the somatodendritic domain in virtually all neurons. However, FMRP is also localized in discrete granules (Fragile X granules; FXGs) in a subset of brain regions including frontal cortex, hippocampal area CA3 and olfactory bulb glomeruli. Immunoelectron microscopy shows that FMRP is localized at presynaptic terminals and in axons within these FXG-rich regions. With the exception of the olfactory bulb, FXGs are prominent only in the developing brain. Experiments in regenerating olfactory circuits indicate that peak FXG expression occurs 2–4 weeks after neurogenesis, a period that correlates with synapse formation and refinement. Virtually all FXGs contain FXR2P, while region-selective subsets harbor FMRP and/or FXR1P. Genetic studies show that FXR2P is essential for FXG expression, while FMRP regulates FXG number and developmental profile. These findings suggest that Fragile X proteins play a distinct, presynaptic role during discrete developmental epochs in defined circuits of the mammalian CNS. We propose that the neurological defects in Fragile X syndrome, including the autistic features, could be due in part to the loss of FMRP function in presynaptic compartments.

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.

Building synapses: agrin and dystroglycan stick together

A major effort of the past decade for those studying synaptic development has been to identify the molecular signals whose carefully choreographed exchange between pre- and postsynaptic cells regulates the local differentiation of each cell to form the mature synapse. Now that several of these factors [agrin, ACh-receptor inducing activity (ARIA) and calcitonin gene-related peptide] have been identified and isolated, efforts have moved toward understanding their receptors and the intracellular signaling pathways by which the factors achieve their effects. One of the most intensively studied of the synaptic signaling molecules is agrin, a large protein synthesized and released by motor neurons that induces ACh receptors and other synaptic molecules in muscle cells to accumulate at the sites of nerve contact. Recent efforts to discover the agrin receptor have led to a surprising conclusion: the only agrin-binding component so far detected in muscle cells is dystroglycan, an extracellular protein that is part of the complex of proteins associated with dystrophin, and its homologue, utrophin. Because dystroglycan binds laminin, and dystrophin binds actin, the complex containing these two proteins is thought to link the extracellular matrix to the cytoskeleton. Those interested in synapses are now pondering whether dystroglycan has a new and unexpected role as a signaling receptor for agrin-induced ACh-receptor clustering, whether it serves as an auxiliary for another receptor, or whether it serves as a receptor for an entirely different agrin-mediated function.

Biglycan recruits utrophin to the sarcolemma and counters dystrophic pathology in mdx mice

Duchenne muscular dystrophy (DMD) is caused by mutations in dystrophin and the subsequent disruption of the dystrophin-associated protein complex (DAPC). Utrophin is a dystrophin homolog expressed at high levels in developing muscle that is an attractive target for DMD therapy. Here we show that the extracellular matrix protein biglycan regulates utrophin expression in immature muscle and that recombinant human biglycan (rhBGN) increases utrophin expression in cultured myotubes. Systemically delivered rhBGN up-regulates utrophin at the sarcolemma and reduces muscle pathology in the mdx mouse model of DMD. RhBGN treatment also improves muscle function as judged by reduced susceptibility to eccentric contraction-induced injury. Utrophin is required for the rhBGN therapeutic effect. Several lines of evidence indicate that biglycan acts by recruiting utrophin protein to the muscle membrane. RhBGN is well tolerated in animals dosed for as long as 3 months. We propose that rhBGN could be a therapy for DMD.

Visual Experience Regulates Transient Expression and Dendritic Localization of Fragile X Mental Retardation Protein

Fragile X syndrome is the most common form of inherited mental retardation and is caused by the loss of function of the Fragile X mental retardation protein (FMRP). FMRP is an RNA binding protein thought to play a key role in protein synthesis-dependent synaptic plasticity. The regulation of FMRP expression itself is also likely to be an important control point in this process. Here we used dark-reared/light-exposed rats to determine the role of experience in regulating FMRP levels in the visual cortex. We find that FMRP levels increase in the cell bodies and dendrites of visual cortical neurons after as little as 15 min of light exposure. Remarkably, FMRP expression in these neurons returns to baseline levels by 30 min of light exposure. These changes were post-transcriptional because the FMR1 mRNA levels remained constant over this time period. A transient increase in FMRP levels was also observed in synaptic fractions prepared from visual cortices of light-exposed animals. In contrast, α-calcium/calmodulin-dependent kinase II expression showed a sustained upregulation under these conditions. Finally, the increase in FMRP expression was inhibited by blockade of NMDA receptors. This tight temporal-spatial regulation suggests that FMRP plays a dynamic role in a distinct epoch of experience-dependent synaptic plasticity.