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Comparative Study
. 2006 Mar 1;26(9):2413-8.
doi: 10.1523/JNEUROSCI.3680-05.2006.

Fragile X Mental Retardation Protein Shifts Between Polyribosomes and Stress Granules After Neuronal Injury by Arsenite Stress or in Vivo Hippocampal Electrode Insertion

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Free PMC article
Comparative Study

Fragile X Mental Retardation Protein Shifts Between Polyribosomes and Stress Granules After Neuronal Injury by Arsenite Stress or in Vivo Hippocampal Electrode Insertion

Soong Ho Kim et al. J Neurosci. .
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Abstract

Fragile X mental retardation protein (FMRP), the lack of which causes fragile X syndrome, is an RNA-binding protein encoded by the FMR1 gene. FMRP accompanies mRNAs from the nucleus to dendritic regions and is thought to regulate their translation at synapses. It has been shown that FMRP moves into nontranslating stress granules (SGs) during heat stress of cultured fibroblasts (Mazroui et al., 2002). We used a novel method to isolate SGs from neurons by virtue of their TIA-1 (T-cell intracellular antigen 1) protein component, and found that FMRP moved out of polyribosomes and into SGs subsequent to oxidative stress. We then examined FMRP changes in subcellular localization resulting from mechanically induced neuronal injury by placement of electrodes into the dentate gyrus and the perforant path of the hippocampus in vivo. During the first 10 min after electrode insertion into one hippocampus, FMRP shifted into SGs and away from polyribosomes, in both hippocampi. Although the injury discharge subsided beyond 10 s, FMRP levels in polyribosomes and stress granules did not return to basal levels until 30 min after electrode penetration. Our findings suggest that procedures for in vivo induction of long-term potentiation or long-term depression should incorporate a 30 min rest period after electrode insertion, and indicate that the contralateral hippocampus cannot be considered an unstimulated control tissue.

Figures

Figure 1.
Figure 1.
Characterization of SGs prepared with antibody-coupled magnetic microbeads. A, TEM picture of immunoprecipitated stress granules as well as other types of granules. Immunoprecipitation was performed to an essential component of stress granules, TIA-1. Magnetic microbeads (the size is ∼50 nm) (white arrowheads) selectively bound to TIA-1 granules (SGs) (white arrows) but not to other kinds of granules (transport granules, etc.) (black arrows). Because it is hard to retrieve intact SGs from a μMACS column after the microbeads and the metal column are magnetized, we adopted a conventional magnetic bead separation for TEM procedures using a small PCR tube and washing the sample gently a few times. This washing is less rigorous than using a μMACS column; hence a small number of other granules were expected in the preparation for TEM. For quantitative study of FMRP, granules without microbeads (hence, not stress granules) were washed off of the magnetic columns by complete washing. B, A magnified SG with a microbead. C, Western blot analysis of immunoprecipitated SGs shows that they have FMRP, FXR1, FXR2, and NUFIP, as well as TIA-1, TIAR, S6, PABP, eIF4E, eIF4G, HuR, which are known components of SGs. Some large ribosomal protein (L4, L7a) are present in SGs, but not L3 or L28 (data not shown). D, As a control, immunoprecipitation (IP) with nonimmune goat IgG did not pull down TIA-1 or FMRP. IP for FMRP brought down TIA-1; an isotype control using mouse IgG2a showed almost undetectable amounts of FMRP and TIA-1. A μMACS column itself did not attract TIA-1 or FMRP as shown in “no antibody, no beads” control.
Figure 2.
Figure 2.
FMRP shifts from polyribosomes to SGs after arsenite stress. Arsenite is known to induce formation of stress granules. A, FMRP level in SGs (n = 4 mice for decapitation only; n = 3 mice for DMEM control; n = 5 mice for arsenite). B, FMRP level in polysomes (n = 2 for decapitation only; n = 3 mice for DMEM control; n = 3 for arsenite). “Decap only” samples are control hippocampi which were dissected out as soon as possible after decapitation and frozen on dry ice. “DMEM” indicates incubation for 10 min in DMEM without arsenite. Data are presented as means ± SEM. *p < 0.05.
Figure 3.
Figure 3.
Recordings of multiple cell discharges in the DG and PP. A, Discharges of the injured neurons were recorded from the electrodes inserted into the PP and DG of hippocampus. Mass discharges subsided after 10 s. B, Cross-correlation analysis was performed to see whether the discharges recorded in the DG were influenced by the injury discharges in the PP. This example showed that (1) about one-half of the discharges in the DG are correlated to those in the PP, and (2) the lag time or latency of the spikes between the two sites is consistent with the calculated conduction time of action potentials along PP axons and the time of one synaptic delay (or latency for a presynaptic signal to cross a neurochemical synapse to initiate a postsynaptic signal).
Figure 4.
Figure 4.
Effects of unilateral hippocampal electrode insertion: Western blot analysis of FMRP level in SGs and polyribosomes. Levels of FMRP in stress granules and polyribosomes in two control groups are shown in each panel; the hippocampus was removed for analysis after decapitation without (C1) and with (C2) anesthesia. Ten minutes after PP and DG electrodes were placed in the right hippocampus, FMRP was significantly reduced in polyribosomes (B) and increased in stress granules (A) in the right hippocampus. The FMRP levels in these compartments returned to the baseline after electrodes were left in the right hippocampus for 30 min. A similar pattern of responses occurred in the contralateral hippocampus, which was not directly injured by electrode insertion and placement, but this was not statistically significant (C, FMRP level in SGs of left undamaged hippocampus; D, FMRP level in polyribosomes of same). n = 4 rats each for C1, n = 3 for C2, n = 3 for each duration of electrode placement. Data are presented as means ± SEM. *p < 0.05.

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