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DENDRITIC TRAFFICKING OF BRAIN-DERIVED NEUROTROPHIC FACTOR mRNA AND NEURONAL ATROPHY

By Andrea Colliva

Abstract

Trafficking and local translation in neuronal dendrites of selected transcripts, like brain-derived neurotrophic factor (BDNF) mRNA, is a mechanism underlying synaptic plasticity, spine remodeling and neuronal development. It was previously demonstrated that BDNF mRNA is targeted to dendrites in an activity dependent manner. BDNF has a complex gene structure that in rodents, giving rise to eleven different variants in the 5’ untranslated region (5’ UTRs) which are alternatively spliced to a common coding region (CDS) having either a short or long 3’UTR region due to the presence of two different polyadenylation sites. BDNF variants have a different spatial distribution in neurons, allowing a fine regulation of BDNF expression. It was previously shown that BDNF CDS encodes a constitutive dendritic targeting signal recognized by the RNA binding protein (RBP) Translin. Moreover different exons could act as signals for soma retention or permissive for dendritic targeting of BDNF mRNA. The two 3'UTRs regulates activity dependent targeting of BDNF transcripts however, their mechanism of action is unknown. In this study, we investigated the mechanisms underlying the BDNF mRNA trafficking under normal conditions and in a disease characterized by neuronal atrophy: Rett syndrome. In particular we analyzed if the different 5'UTRs sequences alone were able to modify the sorting of reporter Green Fluorescent Protein (GFP) mRNA expressed in transfected hippocampal rat neurons in vitro. We found that only three variants promote a constitutive dendritic targeting, while other variants display a similar distribution to those of GFP reporter mRNA alone, both in basal or stimulated conditions. Then, we focused our attention on the characterization of the interaction between endogenous BDNF mRNA and two different subsets of RBP families required for BDNF 3'UTR activity dependent targeting: the cytoplasmic polyadenylation element binding proteins (CPEBs), the Embryonic Lethal Abnormal Vision-like proteins (ELAVLs) and the Fragile-X Mental Retardation Protein (FMRP). Possible interactions between RBPs protein family and BDNF mRNA were verified by Cross Linking Immunoprecipitation (CL-IP) assays on brain lysates. Moreover, we assessed the grade of colocalization in vitro by fluorescent in situ hybridization for endogenous BDNF mRNA coupled to immunofluorescence for the different RBPs families. This study confirms the association of these proteins to BDNF transcripts, highlighting an heterogeneous colocalization profile. Since mutations in RBP genes are known to be involved in neurodevelopment disorders, we investigated if a dysregulation in ribonucleoparticles (RNP) was involved in the pathogenic mechanism of Rett syndrome (RTT). RTT is an X-linked neurodevelopmental disorder affecting 1:10.000 females. We used a mouse model for this syndrome to analyze in vitro the distribution of three classes of RNP in neurons: transporting granules, stress granules and processing bodies. We selected Staufen-1, Tia-1 and DCP1a as specific markers to describe RNP distribution, dimension and labeling intensity. Moreover, we characterized the distribution of BDNF mRNA in RTT neurons to assess if a lack of trophic support occurs in this pathology during stressful condition or during neuronal activity. We found that alterations in the composition of the granules are present in this model, suggesting that an altered RNP homeostasis could be involved in the pathological mechanisms of RTT. Finally, we tried to rescue neuronal atrophy in vitro by treating cultured neurons with Mirtazapine, an antidepressant that is known to increase BDNF levels in rats and that was recently demonstrated to rescue deficits in the brain of RTT mouse. Using a well characterized in vitro staging for the study of neuronal atrophy we demonstrated that morphological deficits of RTT neurons were rescued after treatment with Mirtazapine.Trafficking and local translation in neuronal dendrites of selected transcripts, like brain-derived neurotrophic factor (BDNF) mRNA, is a mechanism underlying synaptic plasticity, spine remodeling and neuronal development. It was previously demonstrated that BDNF mRNA is targeted to dendrites in an activity dependent manner. BDNF has a complex gene structure that in rodents, giving rise to eleven different variants in the 5’ untranslated region (5’ UTRs) which are alternatively spliced to a common coding region (CDS) having either a short or long 3’UTR region due to the presence of two different polyadenylation sites. BDNF variants have a different spatial distribution in neurons, allowing a fine regulation of BDNF expression. It was previously shown that BDNF CDS encodes a constitutive dendritic targeting signal recognized by the RNA binding protein (RBP) Translin. Moreover different exons could act as signals for soma retention or permissive for dendritic targeting of BDNF mRNA. The two 3'UTRs regulates activity dependent targeting of BDNF transcripts however, their mechanism of action is unknown. In this study, we investigated the mechanisms underlying the BDNF mRNA trafficking under normal conditions and in a disease characterized by neuronal atrophy: Rett syndrome. In particular we analyzed if the different 5'UTRs sequences alone were able to modify the sorting of reporter Green Fluorescent Protein (GFP) mRNA expressed in transfected hippocampal rat neurons in vitro. We found that only three variants promote a constitutive dendritic targeting, while other variants display a similar distribution to those of GFP reporter mRNA alone, both in basal or stimulated conditions. Then, we focused our attention on the characterization of the interaction between endogenous BDNF mRNA and two different subsets of RBP families required for BDNF 3'UTR activity dependent targeting: the cytoplasmic polyadenylation element binding proteins (CPEBs), the Embryonic Lethal Abnormal Vision-like proteins (ELAVLs) and the Fragile-X Mental Retardation Protein (FMRP). Possible interactions between RBPs protein family and BDNF mRNA were verified by Cross Linking Immunoprecipitation (CL-IP) assays on brain lysates. Moreover, we assessed the grade of colocalization in vitro by fluorescent in situ hybridization for endogenous BDNF mRNA coupled to immunofluorescence for the different RBPs families. This study confirms the association of these proteins to BDNF transcripts, highlighting an heterogeneous colocalization profile. Since mutations in RBP genes are known to be involved in neurodevelopment disorders, we investigated if a dysregulation in ribonucleoparticles (RNP) was involved in the pathogenic mechanism of Rett syndrome (RTT). RTT is an X-linked neurodevelopmental disorder affecting 1:10.000 females. We used a mouse model for this syndrome to analyze in vitro the distribution of three classes of RNP in neurons: transporting granules, stress granules and processing bodies. We selected Staufen-1, Tia-1 and DCP1a as specific markers to describe RNP distribution, dimension and labeling intensity. Moreover, we characterized the distribution of BDNF mRNA in RTT neurons to assess if a lack of trophic support occurs in this pathology during stressful condition or during neuronal activity. We found that alterations in the composition of the granules are present in this model, suggesting that an altered RNP homeostasis could be involved in the pathological mechanisms of RTT. Finally, we tried to rescue neuronal atrophy in vitro by treating cultured neurons with Mirtazapine, an antidepressant that is known to increase BDNF levels in rats and that was recently demonstrated to rescue deficits in the brain of RTT mouse. Using a well characterized in vitro staging for the study of neuronal atrophy we demonstrated that morphological deficits of RTT neurons were rescued after treatment with Mirtazapine

Topics: RNA_trafficking, Dendritic_atrophy, BDNF, Rett_syndrome, Hippocampal_neurons, Hippocampal_neurons, Settore BIO/09 - Fisiologia
Publisher: Università degli Studi di Trieste
Year: 2016
OAI identifier: oai:arts.units.it:11368/2908061
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