The tumor overexpressed gene (TOG) protein is present in RNA granules that transport myelin basic protein (MBP) mRNA in oligodendrocyte processes to the myelin compartment. Its role was investigated by conditionally knocking it out (KO) in myelinating glia in vivo . TOG KO mice have severe motor deficits that are already apparent at the time of weaning. This phenotype correlates with a paucity of myelin in several CNS regions, the most severe being in the spinal cord. In the TOG KO optic nerve <30% of axons are myelinated. The number of oligodendrocytes in the corpus callosum, cerebellum, and cervical spinal cord is normal. In the absence of TOG, the most patent biochemical change is a large reduction in MBP content, yet normal amounts of MBP transcripts are found in the brain of affected animals. MBP transcripts are largely confined to the cell body of the oligodendrocytes in the TOG KO in contrast to the situation in wild type mice where they are found in the processes of the oligodendrocytes and in the myelin compartment. These findings indicate that MBP gene expression involves a post‐transcriptional TOG‐dependent step. TOG may be necessary for MBP mRNA assembly into translation permissive granules, and/or for transport to preferred sites of translation. GLIA 2017;65:489–501

CGG repeats in the 5'UTR of Fragile X Mental Retardation 1 (FMR1) RNA mediate RNA localization and translation in granules. Large expansions of CGG repeats (> 200 repeats) in FMR1, referred to as full mutations, are associated with fragile X syndrome (FXS). Smaller expansions (55-200 repeats), referred to as premutations, are associated with fragile X tremor ataxia syndrome (FXTAS) and fragile X premature ovarian insufficiency (FXPOI). TMPyP4 is a porphyrin ring compound that destabilizes CGG repeat RNA secondary structure. Here we show that exogenous CGG repeat RNA by itself, lacking the FMRP ORF, microinjected into hippocampal neurons is localized in RNA granules and inhibits translation of ARC RNA, which is localized in the same granules. TMPyP4 rescues translation of ARC RNA in granules. We also show that in human premutation fibroblasts with endogenous CGG repeat expansions in the FMR1 gene, translation of ARC RNA is inhibited and calcium homeostasis is disrupted and both phenotypes are rescued by TMPyP4. Inhibition of granule translation by expanded CGG repeats and rescue of granule translation by TMPy4, represent potential pathogenic mechanism and therapeutic strategy, respectively, for FXTAS and FXPOI.

International Journal of Developmental NeuroscienceVolume 47, Issue Part_A p. 101-101 Article ISDN2014_0328: New mechanism for CGG repeat expansion disorders Rene Norman, Corresponding Author Rene Norman n/a@.dne University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this authorElisa Barbarese, Elisa Barbarese University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this authorJohn H. Carson, John H. Carson University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this author Rene Norman, Corresponding Author Rene Norman n/a@.dne University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this authorElisa Barbarese, Elisa Barbarese University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this authorJohn H. Carson, John H. Carson University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this author First published: 05 November 2015 https://doi.org/10.1016/j.ijdevneu.2015.04.275Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume47, IssuePart_AISDN 2014 AbstractsDecember 2015Pages 101-101 RelatedInformation

International Journal of Developmental NeuroscienceVolume 47, Issue Part_A p. 101-101 Article ISDN2014_0327: CNS myelination is inhibited in the absence of the tumor overexpressed gene protein in oligodendrocytes M.J. Maggipinto, Corresponding Author M.J. Maggipinto n/a@.dne University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this authorJ. Ford, J. Ford University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this authorH. Le, H. Le University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this authorJ. Weyman-Tutolo, J. Weyman-Tutolo University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this authorE. Barbarese, E. Barbarese University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this author M.J. Maggipinto, Corresponding Author M.J. Maggipinto n/a@.dne University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this authorJ. Ford, J. Ford University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this authorH. Le, H. Le University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this authorJ. Weyman-Tutolo, J. Weyman-Tutolo University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this authorE. Barbarese, E. Barbarese University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this author First published: 05 November 2015 https://doi.org/10.1016/j.ijdevneu.2015.04.274Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume47, IssuePart_AISDN 2014 AbstractsDecember 2015Pages 101-101 RelatedInformation

Globoid cell leukodystrophy (GLD), or Krabbe disease, is a rare and often fatal demyelinating disease caused by mutations in the galactocerebrosidase (galc) gene that result in accumulation of galactosylsphingosine (psychosine). We recently reported that the extracellular matrix (ECM) protease, matrix metalloproteinase-3, is elevated in GLD and that it regulates psychosine-induced microglial activation. Here, we examined central nervous system ECM component expression in human GLD patients and in the twitcher mouse model of GLD using immunohistochemistry. The influence of ECM proteins on primary murine microglial responses to psychosine was evaluated using ECM proteins as substrates and analyzed by quantitative real-time polymerase chain reaction, immunocytochemistry, and ELISA. Functional analysis of microglial cytotoxicity was performed on oligodendrocytes in coculture, and cell death was measured by lactose dehydrogenase assay. Tenascin-C (TnC) was expressed at higher levels in human GLD and in twitcher mice versus controls. Microglial responses to psychosine were enhanced by TnC, as determined by an increase in globoid-like cell formation, matrix metalloproteinase-3 mRNA expression, and higher toxicity toward oligodendrocytes in culture. These findings were consistent with a shift toward the M1 microglial phenotype in TnC-grown microglia. Thus, elevated TnC expression in GLD modified microglial responses to psychosine. These data offer a novel perspective and enhance understanding of the microglial contribution to GLD pathogenesis.

Fragile X syndrome (FXS) is caused by lack of expression of fragile X mental retardation protein (FMRP), the product of the Fmr1 gene. In many cases FXS is associated with abnormalities in CNS myelination. Although FMRP is expressed in oligodendrocyte progenitor cells and immature oligodendrocytes (OLGs) previous studies have not detected it in mature, myelin-producing OLGs. FMRP represses translation of myelin basic protein (MBP) RNA in vitro and is believed to prevent premature MBP expression in immature OLGs. Lack of FMRP in FXS could lead to premature myelination and/or myelin abnormalities. Here we show that FMRP is expressed in mature, MBP-positive OLGs of rodents and in MBP-positive human OLGs. We confirm that FMRP is a translational repressor of MBP mRNA in vitro, but at concentrations likely too high to be physiologically relevant in vivo. We find MBP expression in cultured Fmr1 KO OLGs to be similar to wild type, and expression of MBP and other myelin proteins in brain homogenates of the Fmr1 KO mouse to be similar to wild type before, during, and after the period of active myelination. These results suggest that while FMRP is expressed in mature OLGs, myelin abnormalities caused by lack of FMRP expression in FXS are not recapitulated in rodents.

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.

Dendritic RNAs are localized and translated in RNA granules. Here we use single-molecule imaging to count the number of RNA molecules in each granule and to record translation output from each granule using Venus fluorescent protein as a reporter. For RNAs encoding activity-regulated cytoskeletal-associated protein (ARC) or fragile X mental retardation protein (FMRP), translation events are spatially clustered near individual granules, and translational output from individual granules is either sporadic or bursty. The probability of bursty translation is greater for Venus-FMRP RNA than for Venus-ARC RNA and is increased in Fmr1-knockout neurons compared to wild-type neurons. Dihydroxyphenylglycine (DHPG) increases the rate of sporadic translation and decreases bursty translation for Venus-FMRP and Venus-ARC RNAs. Single-molecule imaging of translation in individual granules provides new insight into molecular, spatial, and temporal regulation of translation in granules.

Trafficking of mRNA molecules from the nucleus to distal processes in neural cells is mediated by heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 trans-acting factors. Although hnRNP A2/B1 is alternatively spliced to generate four isoforms, most functional studies have not distinguished between these isoforms. Here, we show, using isoform-specific antibodies and isoform-specific green fluorescent protein (GFP)-fusion expression constructs, that A2b is the predominant cytoplasmic isoform in neural cells, suggesting that it may play a key role in mRNA trafficking. The differential subcellular distribution patterns of the individual isoforms are determined by the presence or absence of alternative exons that also affect their dynamic behavior in different cellular compartments, as measured by fluorescence correlation spectroscopy. Expression of A2b is also differentially regulated with age, species and cellular development. Furthermore, coinjection of isoform-specific antibodies and labeled RNA into live oligodendrocytes shows that the assembly of RNA granules is impaired by blockade of A2b function. These findings suggest that neural cells modulate mRNA trafficking by regulating alternative splicing of hnRNP A2/B1 and controlling expression levels of A2b, which may be the predominant mediator of cytoplasmic-trafficking functions. These findings highlight the importance of considering isoform-specific functions for alternatively spliced proteins.

In oligodendrocytes and neurons genetic information is transmitted from the nucleus to dendrites in the form of RNA granules. Here we describe how transport of multiple different RNA molecules in individual granules is analogous to the process of multiplexing in telecommunications. In both cases multiple messages are combined into a composite signal for transmission on a single carrier. Multiplexing provides a mechanism to coordinate local expression of ensembles of genes in myelin in oligodendrocytes and at synapses in neurons.

Tumor overexpressed gene (TOG) protein, encoded by cytoskeleton-associated protein CKAP5, is a microtubule-associated protein that binds to heterogeneous nuclear ribonucleoprotein (hnRNP) A2. hnRNP A2 is an RNA trafficking factor that associates with myelin basic protein (MBP) mRNA. In oligodendrocytes, TOG, hnRNP A2, and MBP mRNA colocalize in granules that assemble in the perikaryon and are transported to the peripheral network of processes that extends from it. MBP accumulates preferentially in the membrane of the medial and distal portions of these cellular processes. MBP expression was reduced when TOG level was lowered by short-hairpin (sh) RNA. The reduction in TOG did not affect overall cell morphology or the assembly, transport, localization, or number of MBP mRNA-containing granules. Reduced levels of TOG did not affect another oligodendrocyte-specific component, myelin oligodendrocyte glycoprotein, which is expressed at the same time as MBP but translated from mRNA localized in the cell body. Expression in a neural cell line of a green fluorescent protein (GFP)–MBP fusion protein derived from a construct containing GFP and the full-length cDNA for the rat 14 kDa MBP was reduced when TOG level was lowered by shRNA treatment. Expression of GFP, derived from GFP mRNA containing the hnRNP A2 binding element of MBP mRNA, was similarly reduced in cells with low TOG levels. These data indicate that TOG is necessary for efficient translation of MBP mRNA and suggest that this role is mediated by its interaction with hnRNP A2.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a trans-acting RNA-binding protein that mediates trafficking of RNAs containing the cis-acting A2 response element (A2RE). Previous work has shown that A2RE RNAs are transported to myelin in oligodendrocytes and to dendrites in neurons. hnRNP E1 is an RNA-binding protein that regulates translation of specific mRNAs. Here, we show by yeast two-hybrid analysis, in vivo and in vitro coimmunoprecipitation, in vitro cross-linking, and fluorescence correlation spectroscopy that hnRNP E1 binds to hnRNP A2 and is recruited to A2RE RNA in an hnRNP A2-dependent manner. hnRNP E1 is colocalized with hnRNP A2 and A2RE mRNA in granules in dendrites of oligodendrocytes. Overexpression of hnRNP E1 or microinjection of exogenous hnRNP E1 in neural cells inhibits translation of A2RE mRNA, but not of non-A2RE RNA. Excess hnRNP E1 added to an in vitro translation system reduces translation efficiency of A2RE mRNA, but not of nonA2RE RNA, in an hnRNP A2-dependent manner. These results are consistent with a model where hnRNP E1 recruited to A2RE RNA granules by binding to hnRNP A2 inhibits translation of A2RE RNA during granule transport.

In neural cells, certain RNAs are targeted to dendrites by a specific RNA trafficking pathway, termed the A2 pathway, mediated by the trans-acting trafficking factor, heterogeneous nuclear ribonucleoprotein (hnRNP) A2, which binds to an 11 nucleotide cis-acting trafficking sequence, termed the hnRNP A2 response element (A2RE). RNAs containing A2RE-like sequences are recognized by hnRNP A2 in the nucleus and exported to the cytoplasm where they assemble into trafficking intermediates, termed granules, which also contain components of the translation machinery and molecular motors (cytoplasmic dynein and conventional kinesin). RNA granules move along microtubules to the cell periphery where they become localized and where the encoded protein is translated. Intracellular trafficking of RNA molecules by the A2 pathway is mediated by a complex system consisting of five different subsystems, approximately 35 different molecules and approximately 45 different molecular interactions. Specificity in the A2 pathway is provided by specific interactions of hnRNP A2 with different molecular partners in different subsystems. Polarity of RNA trafficking is controlled by transitions of trafficking intermediates between different subsystems. Comprehensive understanding of the A2 RNA trafficking pathway will require quantitative analysis of concentrations and diffusion constants for each of the different molecules, on rates and off rates for each of the different interactions, relevant conditional operators controlling specific interactions, and interactions of different subsystems. Once the necessary quantitative data are available, mathematical models for the different RNA trafficking subsystems can be developed using computational platforms such as the 'Virtual Cell'. Here we describe how each of the subsystems in the A2 system functions and how the different subsystems interact to regulate RNA trafficking.

In neural cells, such as oligodendrocytes and neurons, transport of certain RNAs along microtubules is mediated by the cis-acting heterogeneous nuclear ribonucleoprotein A2 response element (A2RE) trafficking element and the cognate trans-acting heterogeneous nuclear ribonucleoprotein (hnRNP) A2 trafficking factor. Using a yeast two-hybrid screen, we have identified a microtubule-associated protein, tumor overexpressed gene (TOG)2, as an hnRNP A2 binding partner. The C-terminal third of TOG2 is sufficient for hnRNP A2 binding. TOG2, the large protein isoform of TOG, is the only isoform detected in oligodendrocytes in culture. TOG coimmunoprecipitates with hnRNP A2 present in the cytoskeleton (CSK) fraction of neural cells, and both coprecipitate with microtubule stabilized pellets. Staining with anti-TOG reveals puncta that are localized in proximity to microtubules, often at the plus ends. TOG is colocalized with hnRNP A2 and A2RE-mRNA in trafficking granules that remain associated with CSK-insoluble tissue. These data suggest that TOG mediates the association of hnRNP A2-positive granules with microtubules during transport and/or localization.

ABSTRACT In neural cells, such as oligodendrocytesand neurons, transport of certain RNAs along microtubules is mediated by the cis-acting A2RE trafficking element and the cognate trans-acting hnRNP A2 trafficking factor. Using a yeast two-hybrid screen we have identified a microtubule associated protein, TOG2, as an hnRNP A2 binding partner. The C-terminal third of TOG2 is sufficient for hnRNP A2 binding. TOG2, the large protein isoform of the tumor overexpressed gene (TOG), is the only isoform detected in oligodendrocytes in culture. TOG co-immunoprecipitates with hnRNP A2 present in the cytoskeleton (CSK) fraction of neural cells, and both co-precipitate with