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. 2008 Jun;14(6):926-39.
doi: 10.1016/j.devcel.2008.04.003.

A direct role for FMRP in activity-dependent dendritic mRNA transport links filopodial-spine morphogenesis to fragile X syndrome

Jason B Dictenberg  1 , Sharon A Swanger, Laura N Antar, Robert H Singer, Gary J Bassell
Affiliations

A direct role for FMRP in activity-dependent dendritic mRNA transport links filopodial-spine morphogenesis to fragile X syndrome

Jason B Dictenberg et al. Dev Cell. 2008 Jun.

Abstract

The function of local protein synthesis in synaptic plasticity and its dysregulation in fragile X syndrome (FXS) is well studied, however the contribution of regulated mRNA transport to this function remains unclear. We report a function for the fragile X mental retardation protein (FMRP) in the rapid, activity-regulated transport of mRNAs important for synaptogenesis and plasticity. mRNAs were deficient in glutamatergic signaling-induced dendritic localization in neurons from Fmr1 KO mice, and single mRNA particle dynamics in live neurons revealed diminished kinesis. Motor-dependent translocation of FMRP and cognate mRNAs involved the C terminus of FMRP and kinesin light chain, and KO brain showed reduced kinesin-associated mRNAs. Acute suppression of FMRP and target mRNA transport in WT neurons resulted in altered filopodia-spine morphology that mimicked the FXS phenotype. These findings highlight a mechanism for stimulus-induced dendritic mRNA transport and link its impairment in a mouse model of FXS to altered developmental morphologic plasticity.

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Figures

Fig. 1
Fig. 1. Fluorescence in situ hybridization (FISH) of hippocampal neurons
(a-d) Neurons from wild-type (WT, right panels) or FMRP-knockout (KO, left panels) were cultured for 10 days and either not stimulated (‘unstim’, upper panels) or stimulated with 50uM DHPG for 15 minutes (‘DHPG’, lower panels). Corresponding histograms showing dendritic quantification of FISH experiments are shown to the right. Results for WT (black bars) and KO (white bars) are labeled on the x-axis. Close-up images of dendritic signals are shown to the right (in same order). (a) MAP1b mRNA (n=15-16; *p<0.001 for unstimulated vs. DHPG in WT, P>0.25 for KO). (b) CaMKIIa mRNA (n=15-17; *p<0.01 for WT, p>0.2 for KO). There was no significant difference in CaMKIIa abundance of unstimulated neurons between WT and KO (n=15, p>0.1). (c) Beta-actin mRNA (n=11-13; p>0.2). Scale bars=12um. (d-e) FISH for other mRNA targets of FMRP in both WT (right image panels) and KO (left image panels). Representative images shown for unstimulated (upper panels; ‘unstim’) and stimulated (lower panels; DHPG, 50uM), and close-up images of dendrites are displayed above the histograms (in same order). Histograms (right) showing dendritic fluorescence quantification are shown for WT (green) and KO (red) using probes to (d) SAPAP4 (n=16-17; p<0.01 for WT, p>25 for KO) and (e) GABA-A receptor delta (n=13-15; *, ***p<0.01 for unstimulated vs. DHPG in WT and for DHPG in WT and KO, **p<0.05 for unstimulated vs. DHPG in KO). Scale bars=12um. Values are mean±SEM.
Figure 2
Figure 2. Time-lapse analysis of CaMKIIa reporter mRNA transport in live neurons from WT or Fmr1-KO
Neurons (10DIV) were transfected with a GFP-MS2-CaMKIIa mRNA reporter, and exposed to DHPG (50uM, 15 min.). mRNA movements were imaged over a two-minute interval, with each frame captured every 1.5s. (a) Images (left panels) show the first frame (1) and last frame (80) of the time-lapse series of GFP-MS2-CaMKIIa (green) and highlight the tracked mRNA particles (red). The mean-squared displacement (MSD) of individual particle trajectories were analyzed (graph, right) in WT (upper panels) and KO (lower panels) neurons, and an example of the MSD analysis graphs showing 24 particle trajectories for each genotype are shown at right (24 trajectories on graph; many are overlapping at bottom of graph and obscured by icons). The slope of the individual trajectories approximates the particle velocity, and the MSD measures the trajectory length over time. (b) Histogram showing the average number of mRNA particles among several movies (n=6 neurons, 307 particles total, *p<0.02, mean±SEM) from both WT and KO neurons that were motile. (c) Colocalization of CaMKIIa 3′UTR-MS2-GFP with endogenous FMRP. Fluorescence images of a hippocampal dendrite showing MS2-GFP (i, green), CaMKIIa-MS2 reporter mRNA (ii, red) and FMRP (iii, blue). Arrowheads show three granules in dendrites of this representative neuron triple-labeled where all colocalize, which was determined using 3D deconvolution and image reconstruction (see Suppl. Fig. 1f). Scale bar = 5um.
Fig. 3
Fig. 3. The C-terminus of FMRP is involved in kinesin-dependent stimulus-induced transport
(a) Rapid FMRP-GFP movements in live hippocampal neurons (10DIV). (Images) 5 consecutive image panels (top to bottom, left side) show time points (seconds, red) indicated in the upper-right corner and a green arrow tracing the particle movement. (Histograms) Individual particle velocities (top 2 histograms) were traced on a frame-per-frame basis (granule velocities, um/sec, + is anterograde, - is retrograde). Average velocities (bottom histogram) were measured for multiple granules (lower graph, right; n=8, mean values shown). (b) (Upper panel) Western showing KHC and FMRP coIP from cortical cultures. Either nothing (no stim) or DHPG was added to cultures for 15 min and then KHC IP performed. Supts (S) and pellets (P) were analyzed by SDS-PAGE. Arrowhead denotes FMRP band only seen in IP of KHC (other bands in SUPT are likely cross-reacting fragile X related proteins or post-translationally modified FMRP) (Lower Panel) Super-resolution 3-D colocalization analysis of kinesin heavy chain and FMRP in dendrites. Hippocampal neurons were cultured (12DIV), fixed and stained for FMRP (green) and either Kif5 (KHC, left two panels, red) or Kif3 (right two panels, red), and IF images were captured and processed for deconvolution. Images are maximum projections of 3-D stacks, and white pixels represent overlap of the two signals. The histogram shows the average Pearson's coefficient of correlation for each antigen pair as calculated (green bar is for KHC/FMRP and red bar is for Kif3/FMRP; n=14, p<0.0005, mean±SEM). Scale bar, 2um. (c) Domain analysis of FMRP for kinesin interaction. (Top (first) panel - supernat) Quantitative western blot of KLC (anti-HA) shows the supts after the immunoprecipitation (IP) of FMRP domains, with both full-length (FL)-FMRP and C-terminal domain of FMRP (D3, aa386-585) able to significantly deplete the supt compared to the control (CON, no FMRP protein), N-terminal domain (D1, aa1-208) or the central domain (D2, aa290-387) of FMRP. (Second panel- pellet) Western blot against HA shows pellets contain KLC-HA that associate with FMRP by coIP. (Third panel-FMRP pellets) Western blot against FLAG shows the control (CON, no FMRP protein expressed) or IP FL-, D1-, D2-, or D3-FMRP proteins that were used to pull-down KLC-HA. (Histogram) HA-KLC bands pulled-down for each pellet and the corresponding supt were digitally quantified using the LiCOR quantitative blot system and expressed as a ratio of pellet to supt for each FMRP domain. (d) Hippocampal neurons (7DIV) showing FRAP of FMRP-GFP in response to DHPG. (Left Panel) Images of dendrites subjected to FRAP: Upper series show a low magnification image of a control cell (co-transfected KLC-FL) and the dendritic area bleached (inset box), a higher mag image of the subregion just before bleach (B4, second panel) and after (third panel), 1 minute (fourth panel) and 5 minutes later (fifth panel). Lower series show the same sequence for KLC-TPR co-transfected with FMRP-GFP. Scale bar=15um. (Right panel) Graph showing the percent recovery (y-axis) measured over 300 seconds (x-axis) is shown. Squares represent the KLC-TPR transfected cells while the triangles represent FL-KLC (n=6; p<0.005 at 300 second time point). Calculated time constants of recovery (τ, 50%) are shown on the graph. (e) (Upper panels) Representative images showing CaMKIIa mRNA FISH for neurons transfected with either FL-KLC (left) or TPR-KLC (right) constructs and stimulated with DHPG for 15 minutes. Scale bar=15um. (Lower panel) Histogram of CaMKIIa mRNA dendritic abundance in KLC transfected neurons (n=17-22; *p<0.02; mean±SEM). Images to right show close-ups of dendrites from upper-panel images. (f) (Upper panels) Representative images showing MAP2 mRNA FISH for neurons transfected with either FL-KLC (upper) or TPR-KLC (lower) constructs and stimulated with DHPG for 15 minutes. Scale bar=15um. (Lower panel) Histogram of MAP2 mRNA dendritic abundance in KLC transfected neurons (n=12-14; p>0.22; mean±SEM. Images to right show close-ups of MAP2 mRNA in dendrites from upper panel images.
Fig. 4
Fig. 4. FMRP target mRNAs associated with kinesin
Immunoprecipitation (IP) of KHC was used to analyze associated mRNAs by PCR and determine differences between WT and FMRP KO brain. (a) (Upper panel) Ethidium gel for semi-quantitative RT-PCR products from KHC and FMRP IP pellets using primers for beta-actin mRNA (lower bands) and SAPAP4 mRNA (upper bands). ‘Input’ is the input lysate showing starting material for both WT and KO brain. IPs of both kinesin heavy chain (KHC) and FMRP are indicated above the gel, and genotype is indicated below. FMRP IP in KO brain was used to subtract background from other bands, and was ∼4% of WT IP. (Middle panel) Western blot of protein pellets showing that the KHC IPs are identical from WT and KO brain. FMRP antibodies (anti-7G1) also reveal that FMRP is able to precipitate in WT but not KO brain (Lower panel - histogram) Quantification of mRNA bands from PCR (upper panel) by digital fluorescence analysis. (b) Real-time Q-PCR of indicated mRNAs associated with KHC by IP, expressed as a percent of mRNA isolated from KHC in KO compared to WT brain (n=8; *p<0.05; **p<0.01; ***p<0.005; ****p<0.0001; mean±SEM). (c) Real-time Q-PCR analysis of mRNA abundance. Histogram showing that several mRNA targets of FMRP were analyzed for total mRNA levels in both WT (green bars) and KO (red bars) brain derived from P7 mice (n=8, p≥0.4 (minimum)).
Fig. 5
Fig. 5. FMRP and target mRNA transport regulates the length and number of dendritic filopodia-spine protrusions
(a, b) Overexpression of the C-terminal domain (D3) of FMRP in hippocampal neurons. (a) Cultured neurons (11 DIV) were transfected with constructs bearing either FL-FMRP (upper panels) or the C-terminal domain (D3, lower panels) as FLAG-tagged proteins along with FMRP-GFP as a reporter for dendritic transport. GFP fluorescence (left panels) and MAP2 staining (right panels) are shown for representative images. The histogram (below) indicates the average fluorescence intensities of FMRP-GFP in dendrites of treated cells (n=20-23; *p<0.002), with images showing blow-up regions of corresponding dendritic FMRP-GPF outlined in the whole cell images (above). Scale bar = 15uM. (b) Staining of endogenous FMRP after overexpression of D3-FMRP (with CFP as marker). Neurons were stained for endogenous FMRP (red images; using an antibody that does not recognize domain 3 (D3) of FMRP), total RNA (green images; SYTO-Select), and co-transfected CFP is shown (blue). FMRP IF was quantified in dendrites (histogram, below) for both D3 neurons (upper panels, D3/CFP) and control transfected neurons (lower panels, CFP). (n=15-22; *p<0.001). Blow-up images above the histogram bars show the corresponding outlined regions of the whole cell images (above). Scale bar=15um. (c) Dendritic CaMKIIa mRNA localization in response to DHPG (11DIV). Representative images are shown (left) for MAP2 staining (blue) and CaMKIIa mRNA FISH (green) for both D3 (upper panels) and FL FMRP (lower panels) overexpression. Histogram (right) shows quantification of dendritic fluorescence of CaMKIIa (n=13; *p<0.01), with blow-up images above bars showing dendritic regions outlined in the whole cell images (above). Scale bar=10um. (d) Dendritic SAPAP4 mRNA localization in response to DHPG (11DIV). Representative images are shown (left) for MAP2 staining (blue) and SAPAP4 mRNA FISH (green) for both D3 (lower panels) and FL FMRP (upper panels) overexpression. Histogram (right) shows quantification of dendritic fluorescence of SAPAP4 (n=14-15; *p<0.001), with blow-up images above bars showing outlined dendritic regions outlined in whole cell images (above). Scale bar=10um. (e, f) Dendritic filopodia-spine protrusion analysis in response to DHPG. (e) Actin-CFP staining (10 DIV) co-transfected with either D3 or FL FMRP highlights dendritic filopodia-spine protrusion morphology. A representative neuron with D3 overexpression is shown at left, with blow-up panels from representative dendrites at right for both treatments. Scale bar=5um. (f) Histogram showing quantification of dendritic filopodia-spine protrusion number and lengths in D3 or FL FMRP neurons (n=749-852 protrusions measured; *p<0.005 for # protrusions; **p<0.0001 for protrusion length).
Figure 6
Figure 6. Model for FMRP function in linking stimulus-induced dendritic mRNA transport to local translation at synapses
mGluR activation causes a differential accumulation and subsequent translation of mRNAs important for synapse formation and maturation during development in WT neurons; but in neurons lacking FMRP this selective transport-dependent increase in mRNAs is dysregulated.
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