Investigating Sub-Spine Actin Dynamics in Rat Hippocampal Neurons with Super-Resolution Optical Imaging

Morphological changes in dendritic spines represent an important mechanism for synaptic plasticity which is postulated to underlie the vital cognitive phenomena of learning and memory. These morphological changes are driven by the dynamic actin cytoskeleton that is present in dendritic spines. The study of actin dynamics in these spines traditionally has been hindered by the small size of the spine. In this study, we utilize a photo-activation localization microscopy (PALM)–based single-molecule tracking technique to analyze F-actin movements with ∼30-nm resolution in cultured hippocampal neurons. We were able to observe the kinematic (physical motion of actin filaments, i.e., retrograde flow) and kinetic (F-actin turn-over) dynamics of F-actin at the single-filament level in dendritic spines. We found that F-actin in dendritic spines exhibits highly heterogeneous kinematic dynamics at the individual filament level, with simultaneous actin flows in both retrograde and anterograde directions. At the ensemble level, movements of filaments integrate into a net retrograde flow of ∼138 nm/min. These results suggest a weakly polarized F-actin network that consists of mostly short filaments in dendritic spines

Multiplexed dendritic targeting of RNAs by the A2 pathway in hippocampal neurons

In neurons, multiple RNAs are targeted to dendrites. Targeting and local translation of these RNAs is an important mechanism for cytoplasmic gene regulation that modulates synaptic plasticity. It is not known if different dendritically targeted RNAs are localized current knowledge about RNA localization in various systems. The second chapter provides evidence that multiple functionally coherent RNAs (CaMKII, NG and ARC) are localized to dendrites via the heteregenous ribonucleoprotein A2 (hnRNPA2) pathway. A common cis acting element present in these RNAs, called the hnRNPA2 response element (A2RE), interacts with trans acting factor, hnRNPA2 to mediate dendritic targeting. Experiments outlined in the third chapter show that tumor-overexpressed gene (TOG) is a multivalent ligand for hnRNPA2. TOG provides a scaffold for coassembling multiple hnRNPA2 molecules bound to RNA into composite RNA granules. The final step in dendritic RNA targeting is local translation. The fourth chapter describes a novel single molecule assay to measure dendritic translation of RNAs with spatial, temporal and molecular resolution. In the last chapter, we describe the multiplexing hypothesis for dendritic targeting. In multiplexing different dendritically localized RNA are packaged into a composite transport intermediate. Finally, we describe several human that may be caused by perturbing multiplexing.

Upregulation of μ3A Drives Homeostatic Plasticity by Rerouting AMPAR into the Recycling Endosomal Pathway

Synaptic scaling is a form of homeostatic plasticity driven by transcription-dependent changes in AMPA-type glutamate receptor (AMPAR) trafficking. To uncover the pathways involved, we performed a cell-type-specific screen for transcripts persistently altered during scaling, which identified the μ subunit (μ3A) of the adaptor protein complex AP-3A. Synaptic scaling increased μ3A (but not other AP-3 subunits) in pyramidal neurons and redistributed dendritic μ3A and AMPAR to recycling endosomes (REs). Knockdown of μ3A prevented synaptic scaling and this redistribution, while overexpression (OE) of full-length μ3A or a truncated μ3A that cannot interact with the AP-3A complex was sufficient to drive AMPAR to REs. Finally, OE of μ3A acted synergistically with GRIP1 to recruit AMPAR to the dendritic membrane. These data suggest that excess μ3A acts independently of the AP-3A complex to reroute AMPAR to RE, generating a reservoir of receptors essential for the regulated recruitment to the synaptic membrane during scaling up

Conditional knockout of tumor overexpressed gene in mouse neurons affects RNA granule assembly, granule translation, LTP and short term habituation.

In neurons, specific RNAs are assembled into granules, which are translated in dendrites, however the functional consequences of granule assembly are not known. Tumor overexpressed gene (TOG) is a granule-associated protein containing multiple binding sites for heterogeneous nuclear ribonucleoprotein (hnRNP) A2, another granule component that recognizes cis-acting sequences called hnRNP A2 response elements (A2REs) present in several granule RNAs. Translation in granules is sporadic, which is believed to reflect monosomal translation, with occasional bursts, which are believed to reflect polysomal translation. In this study, TOG expression was conditionally knocked out (TOG cKO) in mouse hippocampal neurons using cre/lox technology. In TOG cKO cultured neurons granule assembly and bursty translation of activity-regulated cytoskeletal associated (ARC) mRNA, an A2RE RNA, are disrupted. In TOG cKO brain slices synaptic sensitivity and long term potentiation (LTP) are reduced. TOG cKO mice exhibit hyperactivity, perseveration and impaired short term habituation. These results suggest that in hippocampal neurons TOG is required for granule assembly, granule translation and synaptic plasticity, and affects behavior

fEPSPs and LTP in CA1 of control and TOG cKO hippocampal brain slices.

<p>A. Infrared differential interference contrast micrograph of a hippocampal slice showing bipolar tungsten electrode (left) and glass recording electrode (right) placement. B – C. Electrical recording of field responses from control (B) and TOG cKO (C) slices at various stimuli intensities. Presynaptic fiber volley and field excitatory postsynaptic potential (fEPSP) are marked by arrows. Input output curves generated from control (n = 7 slices from 5 animals) and TOG cKO (n = 7 slices from 4 animals) plotted using fEPSP amplitudes (left) and rising slopes (right). Error bars represent standard deviation of the mean. D – E. Field responses and input output curves in the presence of GABAA antagonist (GABAzine, 5 μM) for the same specimens as in B and C. Error bars represent standard deviation of the mean. F. Electrical recording of field responses in control and TOG cKO hippocampal slices, in presence of GABAzine (5 μM), during baseline (BL) as well as 5 and 60 minutes after theta burst stimulation (TBS, 10 bursts in 2 sec). G. Rising slopes of fEPSPs during BL, and 5 and 60 minutes after TBS, from control (n = 5 animals) and TOG cKO (n = 4 animals). * denotes significant difference from BL (repeated measures ANOVA followed by Newman-Keuls multiple comparison test, p<0.05). H. Group time-course before and after TBS, normalized to the rising slopes of its corresponding BL. Error bars represent standard deviation of the mean.* denotes significant difference from BL period in control (repeated measures ANOVA followed by Bonferroni's multiple comparison test, p<0.05).</p

TOG knockout in hippocampal neurons.

<p>A. Diagram of the portion of the <i>TOG/CKAP5</i> gene that is targeted for excision, and the targeting construct. The bottom panel shows the out-of-frame deletion after <i>Cre</i> excision. B. Western blot of brain homogenates from +/+ (control) and +/null mice stained with rabbit anti-TOG and mouse anti-β-actin; error bars indicate standard deviations; n = 6 animals for each genotype (t-test *p<0.05). C. Western blot of homogenates from hippocampus, cerebellum and cortex of 2 month old control (wild type) and TOG cKO mice stained with rabbit anti-TOG, mouse anti-αCaMKII and mouse anti-β-actin; n = 3 animals for each genotype.</p

TOG expression, granule assembly, granule translation and ARC expression in control and TOG cKO hippocampal neurons in culture.

<p>A. Fluorescence microscopic images of a cultured control neuron co-stained with anti-TOG and Alexa 488 conjugated secondary antibody and Texas-red conjugated phalloidin to label f-actin. A'. High magnification of the dendritic segment identified by 2 asterisks in A and stained with anti-TOG. Scale bar  = 10 μm. B. Fluorescence microscopic images of 2 cultured TOG cKO neurons co-stained with anti-TOG and Alexa 488 conjugated secondary antibody and Texas-red conjugated phalloidin. B'. High magnification of the dendritic segment identified by 2 asterisks in B and stained with anti-TOG. Scale bar  = 10 μm. C – F. Subcellular distribution of microinjected Venus-ARC RNA (labeled with Cy5-UTP) in control (C) and TOG cKO (D) hippocampal neurons and in TOG cKO neurons co-injected with full-length recombinant TOG protein (E) or with an equal molar mixture of individual TOG domains [D1–D7] (F). Injected cells were visualized by wide field fluorescence microscopy. Scale bar for C – F = 10 μm. G – J. Number of translation events per 10 sec is plotted versus time. Representative translation profiles under conditions described in C – F. K. Numbers of Venus-ARC RNA containing granules were counted in 10 μm dendritic segments of neurons treated as in C – F. Values represent average and standard deviations for numbers of granules per 10 μm dendritic segments (t-test, *p<0.05). L. Numbers of translation events per burst were counted for individual granules in control, TOG cKO, TOG cKO plus full length TOG protein and TOG cKO plus TOG domains in cells injected with Venus-ARC RNA (labeled with Cy5-UTP). A burst is defined as a sustained period of elevated translation activity (>3 events/10 sec) preceded and followed by periods of lower translation activity (<2 events/10 sec). Values represent average and standard deviations for numbers of events per burst in different granules (t-test, *p<0.05). M – N. ARC protein levels were measured in control and TOG cKO neurons (n = 8) after immunostaining with anti-ARC and a fluorescent secondary antibody. Error bars indicate standard deviations (t-test; *p<0.05). O. Quantification of total (T) and surface (S) GluA1 in control and TOG cKO hippocampal neurons in culture using biotinylation and western blotting. Error bars indicate standard deviations (t-test; *p<0.05). P. Control and TOG cKO hippocampal neurons were stained with anti-Glu A1 after 18 days in culture. The insets in P are low magnification of the same cells stained for actin. Scale bar in M and P = 20 μm.</p