5A,black bars), despite the lower cellular concentration of the tethered protein (Fig. construct, Htt down-regulated reporter gene expression in a manner dependent on Ago2, suggesting that Htt may function to repress translation of mRNAs during transport in neuronal granules. Keywords:Gene Silencing, Neurodegeneration, P-body, RNA Silencing, RNA Transport, Huntington Disease, Neuronal RNA Granules, Post-transcriptional Gene Silencing == Introduction == Transport and local translation of mRNAs in neurons play key Rabbit Polyclonal to Doublecortin (phospho-Ser376) functions in modulating synaptic strength and maintaining proper neural circuitry (1,2). Neuronal RNA granules serve as sites of transport and translational repression of dendritic mRNAs. Several types of cytoplasmic RNA granules in neurons have been described that contain distinct as well as shared components (3). Processing (P)-bodies6are dynamic assemblies of RNA and proteins found in the cytoplasm of somatic cells (4). In response to stress, P-bodies form to store mRNAs targeted for degradation or translational control. It was recently reported that neuronal ribonucleoprotein particles inDrosophilathat contain RNA-binding proteins Staufen and fragile X mental retardation protein are related to somatic P-bodies in structure and function (5). Further, P-body-like structures have been described in mammalian dendritic neurons that are heterogeneous in composition and respond to neuronal activity (6,7). Somatic P-bodies also serve as sites of microRNA (miRNA)-mediated translational repression. Given the similarities between neuronal ribonucleoprotein particles and somatic P-bodies, it has been proposed that miRNAs may help to maintain silencing of mRNAs during transport by neuronal RNA granules. We recently reported that Huntington disease (HD) protein huntingtin (Htt) associates with Argonaute proteins, localizes to P-bodies, and contributes to post-transcriptional gene silencing (8). HD is usually a dominantly inherited late-onset progressive neurodegenerative disorder caused by an growth of CAG trinucleotide repeats, resulting in a long tract of polyglutamines in the N terminus of Htt whose one or more normal functions remain unclear. Mouse models of HD have provided evidence for a disease mechanism Dioscin (Collettiside III) that involves a gain of function of the mutant HD protein (912). Many studies have focused on deciphering the pathogenic mechanisms involving mutant Htt; however, new studies point to a role for wild-type Htt in the disease process (13). Determining the molecular function of wild-type Htt may show crucial to Dioscin (Collettiside III) understanding HD pathogenesis and the eventual development of effective treatment strategies. Htt has been reported to function in retrograde transport of vesicles in neurons; it is required for transport in axons at rates consistent with microtubule-associated vesicles (14,15). Huntingtin-associated protein 1 (HAP1) was identified in a yeast two-hybrid screen using the N-terminal 230 amino acids of Htt as bait (16). HAP1 interacts with p150Glued, a subunit of the vesicular motor protein complex dynein, and HAP1 may act as a link between the motor proteins and their cargo (14). Htt has been proposed to affect vesicle mobility or cargo binding affinity. Overexpression and knockdown assays showed that Htt plays an important role in controlling brain-derived neurotrophic factor trafficking (17); however, trafficking of Htt itself has not been reported. In this study we present findings that implicate Htt in RNA transport and local translation through neuronal RNA granules. == EXPERIMENTAL PROCEDURES == == == == == == Dissection and Culture of Neurons == Rat neurons were isolated and cultured as previously described (18,19). All rats were maintained under veterinary supervision at the New York University School of Medicine Animal Care Facility in accordance with the guidelines established by the National Institutes of Health (NIH) for the care of laboratory animals, and all procedures were approved by the Institutional Animal Care and Use Committee. To prepare brain slices for immunostaining and FISH, wild-type Wistar rats (7 days aged) were perfused transcardially with PBS (pH 7.4), followed by 4% paraformaldehyde under deep anesthesia induced by intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (25 mg/kg). Brains were extracted from the skull and fixed with 4% paraformaldehyde overnight at 4 C. The hippocampus was then dissected out, and 100-m Vibratome sections were prepared with a Vibratome Series 1000 Classic (Vibratome Co., St. Louis, MO) and transferred into 24-well plates filled with PBS. All dilutions and washes (3 30 min) between stages were performed in PBS. Vibratome sections were washed for 20 min with Dioscin (Collettiside III) PBS, blocked with 5% goat serum (Sigma) for 2 h, and incubated overnight with mouse -Htt and rabbit -Dcp1 antibody (in answer made up of 5% goat serum) at 4 C. Sections were then incubated overnight with Alexa 488-conjugated goat.