1. The Retromer Supports AMPA Receptor Trafficking During LTP
Paul Temkin, Wade Morishita, Debanjan Goswami, Kristin Arendt, Lu Chen, Robert Malenka 
Neuron 
Volume 94, Issue 1, Pages e5 (April 2017)
DOI: /j.neuron
Copyright © 2017 Elsevier Inc. Terms and Conditions

2. Figure 1 Retromer Depletion Specifically Blocks LTP
(A) Representative image from hippocampal culture showing one cell (1) infected with lentivirus expressing the VPS35-shRNA-KD construct and GFP (construct diagram above) and a neighboring uninfected cell (2). Image at left shows GFP (green) and VPS35 antibody staining (red). Magnified inset boxes at right show early endosome marker EEA1 (grayscale) and VPS35 (red) antibody staining. Scale bar represents 20 μm.
(B) Cropped western blot from uninfected and VPS35-KD hippocampal cultures, probed with VPS35 and GAPDH antibodies. Approximate location of nearest molecular weight markers is shown at right.
(C) Image showing targeting of VPS35-KD lentivirus in mouse hippocampal slice. ∗ represents a typical recording site in the CA1 pyramidal cell layer, while + represents a typical stimulation site. Scale bar represents1 mm.
(D–F) Summary time course (left), cumulative frequency plot of plasticity magnitude for all experiments in the set (middle), and quantification of plasticity magnitude (right) for molecularly manipulated and corresponding interleaved control neurons. (D) NMDAR-dependent LTP; (E) NMDAR-dependent LTD; (F) NMDAR-independent LTP, induced by repetitive L-type Ca2+-channel activation.
Experimental n values are shown in bars and denote cells/animals. All values plotted with error bars are expressed as mean ± SEM. ∗∗∗p <
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3. Figure 2
Retromer Depletion Does Not Affect Basal Synaptic Properties or Levels of Extrasynaptic AMPARs
(A–F) Measurements from uninfected (black) and VPS35 KD (green) CA1 pyramidal neurons in acute hippocampal slices with representative traces shown above. (A) Spontaneous mEPSC frequency; scale bars represent 1 s/20 pA. (B) Spontaneous mEPSC amplitude; scale bars represent 25 ms/5 pA. (C) Input-output relationship for pairs of adjacent infected and uninfected cells. Averaged traces for 10, 30, and 60 μA are shown above. The linear fit slope is graphed at right. Scale bars represent 50 ms/100 pA. (D) Ratio of AMPAR-mediated to NMDAR-mediated EPSCs. Representative EPSCs at −70 mV and +40 mV are shown above. Scale bars represent 25 ms/50 pA. (E) Paired pulse ratios. Normalized representative recordings are shown above. Scale bar represents 100 ms. (F) AMPAR-mediated currents from nucleated somatic patches. Representative recordings are shown above. In (F), black dot denotes time at which AMPA application was initiated for 1 s at 10 μM. Scale bars represent 1 s/500 pA.
(G) Quantification of spine density on secondary dendrites in stratum radiatum. Scale bar represents 1 μm.
Experimental n value denotes cells/animals. All values plotted with error bars are expressed as mean ± SEM. ∗∗∗p <
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4. Figure 3 Retromer Depletion Blocks Exocytosis of AMPAR during LTP
(A) Comparison of effects of NASPM, a blocker of GluA2-lacking AMPARs, on EPSCs recorded from control GluA1-KO hippocampal cultures and from those infected with lentivirus expressing SEP-GluA1. NASPM was applied at time zero. Example EPSCs are shown for 0 and 100 s after NASPM treatment. Scale bars represent 25 ms/50 pA.
(B) Summary time course (left), cumulative frequency plot of LTP magnitude for all experiments in the set (middle), and quantification of LTP magnitude (right) for uninfected cells and SEP-GluA1-expressing cells in slices prepared from GluA1-KO mice.
(C) Representative images (left) and summary graph of normalized fluorescence (right) for cultured hippocampal neurons expressing SEP-GluA1, imaged every 5 min. Cells were photobleached prior to induction of chemical LTP. Scale bar represents 5 μm.
(D) Summary graph of normalized fluorescence for cultured hippocampal neurons expressing transferrin receptor SEP and tdTomato or a VPS35-KD tdTomato construct, imaged every 5 min after photobleaching.
(E) Cultured hippocampal neurons infected with SEP-GluA1 containing an N-terminal FLAG epitope were fed M1 anti-FLAG antibody, which binds in a Ca2+ dependent manner, for 2 hr, and surface binding was stripped using PBS EDTA. Images of internal SEP-GluA1 and EEA1 are shown at left. Scale bar represents 5 μm. Quantification of percent of internal SEP-GluA1 contained in EEA1-stained region is shown at right.
(F and G) Quantification of EEA1 puncta size (F) and density (G) in primary dendrites of cultured hippocampal neurons.
(H) Model of hypothesized role for retromer in sorting of AMPARs into vesicles for delivery to plasma membrane following LTP induction.
Experimental n values denote cells/cultures for A and C and cells/animals for B. All values plotted with error bars are expressed as mean ± SEM. ∗∗p < 0.005, ∗∗∗p <
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5. Figure 4 Function of VPS35 Disease Mutants in LTP
(A) Summary time course (left), cumulative frequency plot of LTP magnitude for all experiments in the set (middle), and quantification of LTP magnitude for uninfected control cells and VPS35 KD cells in slices prepared from APP-KO mice.
(B–D) Representative images of VPS35-mRuby rescue constructs expressed in cultured hippocampal neurons. Images show section of primary dendrite stained with early endosome marker EEA1. Construct maps are shown above image set. Scale bar represents 5 μm. (B) Wild-type VPS35 construct. (C) A familial Parkinson’s disease mutation, VPS35 D620N. (D) A spontaneous mutation, VPS35 L625P, found in an Alzheimer’s patient. (E) LTP is rescued in VPS35-KD cells by expression of VPS35 L625P, but not VPS35 D620N. Two data points from the “Uninfected” cells are not shown in the cumulative frequency plot (406 and 540).
Experimental n value denotes cells/animals. All values plotted with error bars are expressed as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.
Neuron  , e5DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions