Quantitative proteomic analysis of primary neurons reveals diverse changes in synaptic protein content in fmr1 knockout mice

Abstract

Fragile X syndrome (FXS) is a common inherited form of mental retardation that is caused, in the vast majority of cases, by the transcriptional silencing of a single gene, fmr1. The encoded protein, FMRP, regulates mRNA translation in neuronal dendrites, and it is thought that changes in translation-dependent forms of synaptic plasticity lead to many symptoms of FXS. However, little is known about the potentially extensive changes in synaptic protein content that accompany loss of FMRP. Here, we describe the development of a high-throughput quantitative proteomic method to identify differences in synaptic protein expression between wild-type and fmr1؊/؊ mouse cortical neurons. The method is based on stable isotope labeling by amino acids in cell culture (SILAC), which has been used to characterize differentially expressed proteins in dividing cells, but not in terminally differentiated cells because of reduced labeling efficiency. To address the issue of incomplete labeling, we developed a mathematical method to normalize protein ratios relative to a reference based on the labeling efficiency. Using this approach, in conjunction with multidimensional protein identification technology (MudPIT), we identified >100 proteins that are up-or down-regulated. These proteins fall into a variety of functional categories, including those regulating synaptic structure, neurotransmission, dendritic mRNA transport, and several proteins implicated in epilepsy and autism, two endophenotypes of FXS. These studies provide insights into the potential origins of synaptic abnormalities in FXS and a demonstration of a methodology that can be used to explore neuronal protein changes in neurological disorders. stable isotope labeling ͉ proteomics ͉ mass spectrometry ͉ fragile X syndrome F ragile X syndrome (FXS) is the most common inherited form of mental retardation. It is characterized by low IQ (1) and a broad set of symptoms other than retardation that compound the level of impairment. These include autistic spectrum behaviors, attention deficit and hyperactivity, childhood seizures, and several physical manifestations (2, 3). The most profound neuroanatomical abnormality seen in the brains of FXS patients is a preponderance of long, thin, and ''tortuous'' dendritic spines in cortex (4). This cortical phenotype is recapitulated in the fmr1 knockout (KO) mouse model, which also exhibits abnormal spine morphologies in the hippocampus and cerebellum (5, 6).In the vast majority of cases, FXS is caused by expansion of a trinucleotide repeat (CGG) within the 5Ј-untranslated region of the X-linked gene fmr1, resulting in its transcriptional silencing. The encoded protein, FMRP, can act as a translational suppressor of mRNAs in dendrites, controlling the localized, activity-dependent expression of a potentially large subset of synaptic proteins (7). Knocking out fmr1 in mouse results in a perturbation of various forms of translation-dependent synaptic plasticity, including some forms of long-term potentiation (LTP) and group I metabotropic...

Fmr1 deficiency promotes age-dependent alterations in the cortical synaptic proteome

“…The 15 N-enriched mouse brain homogenates were then mixed at a 1:1 protein ratio with cortical homogenates from either Fmr1 KO or wild-type mice. Crude synaptosomes were prepared according to previously published protocol (19). Briefly, the homogenates were centrifuged at 700 × g for 15 min, followed by centrifuging the resulting supernatant fractions (S1) at 10,000 × g for 15 min.…”

“…Such complexities can be captured only by direct quantification of changes at the protein level. Prior work by our group and others used proteomic methods to identify proteome changes in synaptic fractions from Fmr1 knockout (KO) primary neurons and in dFmr1 Drosophila (19,20). However, these studies likely underestimated the extent of proteome shifts caused by loss of FMRP.…”

“…To further differentiate the role of de novo translation in protein changes from alterations in other processes such as mRNA stability, protein degradation, or trafficking, stable isotope pulsed labeling (pSILAC) has been developed to measure genome-scale protein synthesis rates (27). However, applying pSILAC in cultured primary neurons has not been implemented, presumably due to the low protein turnover in this cell type (19). With these combined approaches, we define a set of nearly 1,000 proteins that are differentially expressed in Fmr1 cortical synapses at P17, including a diverse array of proteins involved in synaptic development and function, metabolism, and a large number corresponding to FMRP HITS-CLIP targets and autism susceptibility genes.…”

Quantitative Nanoproteomics for Protein Complexes (QNanoPX) Related to Estrogen Transcriptional Action

“…In principle, all the components, even of large complexes, can be identified in a single LC-MS experiment. Furthermore, quantitative proteomics approaches that are based upon stable isotope labeling, when performed along with appropriate control experiments, can distinguish background contamination or nonspecific binding from true interactors or differentiating effects that are caused by different biological states (7)(8)(9). Improvements in affinity purification that can be coupled with quantitative proteomics have also been developed, and most of these methods focus on the use of single/dual affinity tags (10,11) or chemical reactions (12), such as the use of in vivo cross-linking agents.…”

La protéomique quantitative par la méthode SILAC

“…Géné-ralement, après 6 à 8 temps de doublement de la population cellulaire, toutes les protéines ont incorporé les acides aminés alourdis avec un taux d'incorporation proche de 100 %. Il a récemment été démontré que la méthode SILAC n'était pas restreinte aux cellules qui prolifèrent en culture, mais pouvait être appliquée à des cellules neuronales primaires [21,22]. Dans le cas de « souris SILAC », le marquage total de toutes les protéines a été obtenu au cours de la deuxième génération de souris nourries avec de la lysine marquée 13 C [19].…”