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

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