Single molecule analysis of AMPAR mRNA expression and localization under physiological and pathological conditions
Fragile X Syndrome is the most prevalent inherited form of intellectual disability and autism and is caused by the silencing of the FMR1 gene encoding the RNA binding protein Fragile X Mental Retardation Protein (FMRP). The mouse model of Fragile X is an Fmrl knockout mouse. A hallmark feature of Fragile X mice is exaggerated metabotropic glutamate receptor long-term depression (mGluR-LTD) at Schaffer collateral to CA1 pyramidal synapses of the hippocampus. mGluR-LTD is implicated in spatial learning and memory consolidation and can be caused by internalization of AMPARs or, in some cases, a change in AMPAR subunit composition. Whereas a role for activity-dependent local translation and alterations in gene expression are implicated in synaptic plasticity, a role for stimulus-evoked alterations in AMPAR transcription (and nascent mRNAs) in synaptic plasticity are, as yet, unknown.;AMPA receptors (AMPARs) are ionotropic glutamate receptors that play a vital role in fast transmission at excitatory synapses in the central nervous system (CNS) and are critical to neuronal development, synaptic plasticity and structural remodeling. Whereas synaptic plasticity is thought to arise through changes in AMPA receptor phosphorylation status and number, it can also arise owing to a change in AMPAR phenotype from GluA2-containing to GluA2- lacking or GluA2-lacking to GIuA2containing. The GluA2 subunit in heteromeric AMPARs renders the channel impermeable to Ca2+ and Zn 2+ and electrically linear, influences channel kinetics, conductance, AMPAR assembly, forward trafficking from the endoplasmic reticulum (ER) and targeting to and from synaptic sites. Thus, even a modest alteration in the level of GluR2 expression would be expected to have profound implications for synaptic efficacy and neuronal survival.;Previous studies from our laboratory reveal that activation of group 1 mGluRs causes an increase in the abundance of GluA2 mRNA in dendrites. However, the downstream effecter of mGluRs that promotes an increase in GluA2 mRNA abundance is not yet known. The RNA binding protein, cytoplasmic adenylation element binding protein 3 (CPEB3), is a putative target of FMRP, and directly binds GluA2 mRNA at a specific sequence within its 3'UTR. Basally, CPEB3 represses GluA2 translation, whereas application of NMDA releases the brake on GluA2 translation. Whilst CPEB3's role in regulating translation is well established it is unknown whether CPEB3 impacts GluA2 transcription, stability and/or transport.;In Chapter 2, we address the impact of group I mGluR activation on GluA2 mRNA transcription and localization to dendrites in rat hippocampal neurons. We found that mRNAs encoding AMPAR subunits GluA1 and GluA2 mRNA appear as distinct packets or granules with little or no colocalization and GluA1 mRNA is more abundant and stable then GluA2 mRNA at basal levels in hippocampal neurons. We further show utilizing single molecule FISH that activation of group 1 mGluR causes a significant increase in GluA2 mRNA transcription leading to an increase in GluA2 mRNA number in the soma and dendrites. Conversely, mGluR activation decreases the number of GIuA1 mRNAs at transcription sites, soma and dendrites. Furthermore, mGluR stimulation resulted in an elevation in CPEB3 translation, resulting in an elevation in global, dendritic, and nuclear CPEB3. To determine whether the mGluR increase in CPEB3 protein and GluA2 mRNA are causally related we examined the impact of mGluR stimulation after CPEB3 knockdown. We observed that acute knockdown of CPEB3 abolishes the mGluR-induced increase in GluA2 mRNA abundance. In addition, loss of CPEB3 blocked the mGluR dependent internalization of GluA2 protein at the surface. Activation of synaptic (but not extrasynaptic) NMDARs caused a significant elevation in the number of GIuA1 and GIuA2 mRNA at transcription sites and in the soma. Lastly, we observed that Fragile X neurons exhibited elevated numbers of GIuA2 mRNA particles. Blockade of group 1 mGluRs rescued the elevated GIuA2 mRNA in Fragile X neurons. Together this study uncovered a novel mechanism by which activation of mGluRs results in elevated GIuA2 transcription and CPEB3 translation leading to an increase in GIuA2 mRNA in dendrites that are strategically position to be synthesized and inserted into the membrane in response to synaptic input.;In Chapter 3 we examined whether excessive protein synthesis downstream of mGluRs in the mouse model of Fragile X leads to altered GIuA2 mRNA and CPEB3 protein abundance. We show that GIuA2, but not GIuA1, mRNA and CPEB3 protein are elevated in dendrites of hippocampal neurons from Fragile X vs wild-type. Acute knockdown of CPEB3 reduced the elevated number of GIuA2 mRNA in Fragile X dendrites to wild-type levels. To determine whether the elevation of CPEB3 protein was due to changes in transcription, translation or protein stability, we assessed global CPEB3 mRNA and protein abundance and found no change in CPEB3 mRNA, but a significant increase in global CPEB3 protein. Additionally, we found that FMRP and CPEB3 are causally related; acute knockdown of FMRP resulted in elevation of CPEB3 protein abundance and introduction of human FMRP in knockout neurons reduced the elevated CPEB3 in knockout neurons to wild-type levels. Next, to determine whether the changes in GIuA2 mRNA are due transcription, we examined the number of GIuA2 mRNAs at transcription sites and in the soma and found, that similarly to dendrites, there is no change in GluA1 mRNA, but an increase in GluA2 mRNA at transcription sites and in the soma of Fragile X neurons. To examine the impact of CPEB3 on GIuA2 mRNA at transcription sites, and total somatic abundance, we utilized genetic manipulation of CPEB3 in wild-type neurons and observed that acute CPEB3 knockdown greatly reduced the number of GIuA2 mRNA at transcription sites and in the soma. Conversely, over expression of CPEB3 causes an increase in GluA2 mRNA. Furthermore, we observed that acute knockdown of CPEB3 rescued the elevated GluA2 mRNA phenotype in Fragile X neurons. To confirm that CPEB3 is acting at the transcriptional level, we performed chromatin immunoprecipitation and found that CPEB3 protein interacts with the Gria2 gene, encoding GluA2, in the promoter region. This study provides novel evidence at the single molecule level that in FXS there is an elevation of CPEB3 protein and GluA2 mRNA. Because of the importance of AMPARs on synaptic plasticity and the specific impact that increases in GluA2 protein in an AMPAR decrease AMPAR conductance, alteration in AMPAR subunit content in Fragile X could contribute in the exaggerated LTD phenotype associated with impaired learning and memory in this devastating human disorder.
Source: Dissertation Abstracts International, Volume: 77-03(E), Section: B.;Advisors: R. Suzanne Zukin.