Quantitative nucleotide level analysis of regulation of translation in response to depolarization of cultured neural cells

Studies on regulation of gene expression have contributed substantially to understanding mechanisms for the long-term activity-dependent alterations in neural connectivity that are thought to mediate learning and memory. Most of these studies, however, have focused on the regulation of mRNA transcription. Here, we utilized high-throughput sequencing coupled with ribosome footprinting to globally characterize the regulation of translation in primary mixed neuronal-glial cultures in response to sustained depolarization. We identified substantial and complex regulation of translation, with many transcripts demonstrating changes in ribosomal occupancy independent of transcriptional changes. We also examined sequence-based mechanisms that might regulate changes in translation in response to depolarization. We found that these are partially mediated by features in the mRNA sequence—notably upstream open reading frames and secondary structure in the 5′ untranslated region—both of which predict downregulation in response to depolarization. Translationally regulated transcripts are also more likely to be targets of FMRP and include genes implicated in autism in humans. Our findings support the idea that control of mRNA translation plays an important role in response to neural activity across the genome

Evolution of Molecular Function in Mammalian Neurons

A common question in neuroscience is what forms the neurological basis of the variety of behaviors in mammals. While many studies have compared mammalian anatomy and evolution, few have investigated the physiologic functions of individual neurons in a comparative manner. Neurons’ ability to modify their features in response to stimuli, known as synaptic plasticity, is fundamental for learning and memory. A key feature of synaptic plasticity involves delivering mRNA to distinct domains where they are locally translated. Regulatory coordination of these events is critical for synaptogenesis and synaptic plasticity as defects in these processes can lead to neurological diseases. In this work, we combine computational and experimental biology to investigate subcellular localization of mRNAs in dendrites of mouse and rat neurons. Differential subcellular localization of specific gene products may highlight differential synaptic function and allow the uncovering of evolutionary conserved as well as divergent molecular functions in neurons. First, we performed a comparative analysis of the dendritic transcriptome in mouse and rat via microarrays. We found that their dendritic transcriptome are significantly more divergent than other homologous tissues and that these evolutionarily changes could be associated with transposon activity. Second, we comprehensively determined subcellular expression patterns for neuronal genes in mice and rats via a systematic in situ survey on a curated list of dendritic mRNA from our previous microarray study. This survey highlighted that dendritic localization of specific transcripts occurs in a species-specific fashion. We uncovered species-specific cis and trans-elements with possible implications in transcript localization and gene expression regulation in neurons. The interactions between these elements might play a major role in the proper development and evolution of complex nervous systems. Our data will be publically available in a database (http://kim.bio.upenn.edu/insitu/public/), which could guide future investigations. Finally, we investigated single cell variability by combining microarrays, RNAseq and in situ experiments. Our preliminary results underlined the extent of this variability and its contributions in establishing cell’s unique identity. In conclusion, our study suggests the existence of species-specific mRNA localization mechanisms and supports the idea that evolution of phenotypes might be linked to the evolution of subcellular localization of transcripts

Whole transcriptome profiling reveals the RNA content of motor axons

Most RNAs within polarized cells such as neurons are sorted subcellularly in a coordinated manner. Despite advances in the development of methods for profiling polyadenylated RNAs from small amounts of input RNA, techniques for profiling coding and non-coding RNAs simultaneously are not well established. Here, we optimized a transcriptome profiling method based on double-random priming and applied it to serially diluted total RNA down to 10 pg. Read counts of expressed genes were robustly correlated between replicates, indicating that the method is both reproducible and scalable. Our transcriptome profiling method detected both coding and long non-coding RNAs sized >300 bases. Compared to total RNAseq using a conventional approach our protocol detected 70% more genes due to reduced capture of ribosomal RNAs. We used our method to analyze the RNA composition of compartmentalized motoneurons. The somatodendritic compartment was enriched for transcripts with post-synaptic functions as well as for certain nuclear non-coding RNAs such as 7SK. In axons, transcripts related to translation were enriched including the cytoplasmic non-coding RNA 7SL. Our profiling method can be applied to a wide range of investigations including perturbations of subcellular transcriptomes in neurodegenerative diseases and investigations of microdissected tissue samples such as anatomically defined fiber tracts

The axonal transcript Tp53inp2 mediates the development of the sympathetic nervous system

Nerve Growth Factor (NGF) is a neurotrophin essential for the survival of sympathetic and sensory neurons. Localisation of mRNA in axons of NGF- dependent neurons supports growth and maintains axon integrity, however how localised transcripts regulates most axonal functions remains unknown. To characterise the 3’UTR of transcripts localised in sympathetic neuron axons, we performed a 3’end RNA-Seq on mRNA isolated from either axons or cell bodies of neurons cultured in compartmentalised chambers. We identified Tp53inp2 as the most abundant transcripts in axons, accounting for almost one third of the reads. Interestingly, despite the abundance of its RNA, the protein for Tp53inp2 is not detectable within axons of sympathetic neurons. We observe that Tp53inp2 is not actively translated, held in a strictly repressed state mediated by its UTRs. Deletion of Tp53inp2 in sympathetic neurons in vivo and in vitro affects both cell survival and axon growth, suggesting a critical role for Tp53inp2 in neuronal development, despite the lack of translation. That this phenotype can be rescued by transfecting a non- translatable form of the transcript, suggests that instead Tp53inp2 acts as an atypical non-coding RNA, whose function is mediated through interaction with the NGF receptor TrkA. We conclude that Tp53inp2 mRNA regulates sympathetic neuron survival and axon growth in a coding-independent manner by interacting with TrkA receptor and enhancing axonal NGF- dependent signalling

Choice of Alternative Polyadenylation Sites, Mediated by the RNA-Binding Protein Elavl3, Plays a Role in Differentiation of Inhibitory Neuronal Progenitors

Alternative polyadenylation (APA) is a widespread mechanism involving about half of the expressed genes, resulting in varying lengths of the 3′ untranslated region (3′UTR). Variations in length and sequence of the 3′UTR may underlie changes of post-transcriptional processing, localization, miRNA targeting and stability of mRNAs. During embryonic development a large array of mRNAs exhibit APA, with a prevalence of the longer 3′UTR versions in differentiating cells. Little is known about polyA+ site usage during differentiation of mammalian neural progenitors. Here we exploit a model of adherent neural stem (ANS) cells, which homogeneously and efficiently differentiate into GABAergic neurons. RNAseq data shows a global trend towards lengthening of the 3′UTRs during differentiation. Enriched expression of the longer 3′UTR variants of Pes1 and Gng2 was detected in the mouse brain in areas of cortical and subcortical neuronal differentiation, respectively, by two-probes fluorescent in situ hybridization (FISH). Among the coding genes upregulated during differentiation of ANS cells we found Elavl3, a neural-specific RNA-binding protein homologous to Drosophila Elav. In the insect, Elav regulates polyA+ site choice while interacting with paused Pol-II promoters. We tested the role of Elavl3 in ANS cells, by silencing Elavl3 and observed consistent changes in 3′UTR length and delayed neuronal differentiation. These results indicate that choice of the polyA+ site and lengthening of 3′UTRs is a possible additional mechanism of posttranscriptional RNA modification involved in neuronal differentiation

A Upf3b-mutant mouse model with behavioral and neurogenesis defects.

Nonsense-mediated RNA decay (NMD) is a highly conserved and selective RNA degradation pathway that acts on RNAs terminating their reading frames in specific contexts. NMD is regulated in a tissue-specific and developmentally controlled manner, raising the possibility that it influences developmental events. Indeed, loss or depletion of NMD factors have been shown to disrupt developmental events in organisms spanning the phylogenetic scale. In humans, mutations in the NMD factor gene, UPF3B, cause intellectual disability (ID) and are strongly associated with autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD) and schizophrenia (SCZ). Here, we report the generation and characterization of mice harboring a null Upf3b allele. These Upf3b-null mice exhibit deficits in fear-conditioned learning, but not spatial learning. Upf3b-null mice also have a profound defect in prepulse inhibition (PPI), a measure of sensorimotor gating commonly deficient in individuals with SCZ and other brain disorders. Consistent with both their PPI and learning defects, cortical pyramidal neurons from Upf3b-null mice display deficient dendritic spine maturation in vivo. In addition, neural stem cells from Upf3b-null mice have impaired ability to undergo differentiation and require prolonged culture to give rise to functional neurons with electrical activity. RNA sequencing (RNAseq) analysis of the frontal cortex identified UPF3B-regulated RNAs, including direct NMD target transcripts encoding proteins with known functions in neural differentiation, maturation and disease. We suggest Upf3b-null mice serve as a novel model system to decipher cellular and molecular defects underlying ID and neurodevelopmental disorders

Loss of the miR379-410 cluster in mice leads to alterations in social and anxiety-related behaviours

microRNAs (miRNAs) belong to a group of small non-coding RNAs that down regulates gene expression at the post-transcriptional level. The paternally imprinted placental mammal-specific miR379-410 cluster hosts 38 miRNAs. In the last decade, several members of the cluster have been shown to regulate synapse development and plasticity in mammals. Further, they have been implicated in a variety of diseases, including neurodevelopmental disorders. However, the potential involvement of these miRNAs in the control of complex behaviour in mammals, such as sociability, remains largely unknown. This is an important issue since aberrant synaptic dysfunction is thought to underlie neurodevelopmental diseases, such as autism spectrum disorder (ASD), characterized by deficits in social communication and interaction as well as restricted repetitive behaviour. This study aimed at the characterization of a constitutive knock-out (ko) mouse model that carries a deletion of the miR379-410 cluster. Extensive behavioural assays across the animals’ lifespan and cellular examinations of structural and functional properties of synapses were performed. Furthermore, transcriptome sequencing of adult miR379-410 ko hippocampi allowed the validation of potential direct target candidates of the miRNA cluster by using molecular and biochemical approaches. Mice deficient for the miR379-410 cluster displayed an anti-autistic-like phenotype, consisting of hypersocial behaviour, increased ultrasonic vocalizations (USVs) and reduced repetitive behaviour in the absence of cognitive impairments. Further, miR379-410 ko mice presented an anxiety phenotype over the lifespan. Along with the behavioural phenotype, miR379-410 ko mice showed increased excitatory synaptic transmission and spine density accompanied by an elevated expression of ionotropic glutamate receptor complex components in the hippocampus. Several of these components, identified by transcriptome profiling (Cnih2, Src, Prr7 and Dlgap3) could be validated as direct miR379-410 target genes. Taken together, the data obtained in this thesis describe for the first time a negative regulatory role of the miR379-410 cluster in social behaviour and the control of genes associated with excitatory synaptic function. Thus, interfering with miRNAs from the miR379-410 cluster could represent in the future a promising strategy for the treatment of neurodevelopmental disorders characterized by social dysfunction, such as ASD

The molecular underpinnings of neuronal cell identity in the stomatogastric ganglion of cancer borealis

Throughout the life of an organism, the nervous system must be able to balance changing in response to environmental stimuli with the need to produce reliable, repeatable activity patterns to create stereotyped behaviors. Understanding the mechanisms responsible for this regulation requires a wealth of knowledge about the neural system, ranging from network connectivity and cell type identification to intrinsic neuronal excitability and transcriptomic expression. To make strides in this area, we have employed the well-described stomatogastric nervous system of the Jonah crab Cancer borealis to examine the molecular underpinnings and regulation of neuron cell identity. Several crustacean circuits, including the stomatogastric nervous system and the cardiac ganglion, continue to provide important new insights into circuit dynamics and modulation (Diehl, White, Stein, & Nusbaum, 2013; Marder, 2012; Marder & Bucher, 2007; Williams et al., 2013), but this work has been partially hampered by the lack of extensive molecular sequence knowledge in crustaceans. Here we generated de novo transcriptome assembly from central nervous system tissue for C. borealis producing 42,766 contigs, focusing on an initial identification, curation, and comparison of genes that will have the most profound impact on our understanding of circuit function in these species. This included genes for 34 distinct ion channel types, 17 biogenic amine and 5 GABA receptors, 28 major transmitter receptor subtypes including glutamate and acetylcholine receptors, and 6 gap junction proteins -- the Innexins. ... With this reference transcriptome and annotated sequences in hand, we sought to determine the strengths and limitations of using the neuronal molecular profile to classify them into cell types. ... Since the resulting activity of a neuron is the product of the expression of ion channel genes, we sought to further probe the expression profile of neurons across a range of cell types to understand how these patterns of mRNA abundance relate to the properties of individual cell types. ... Finally, we sought to better understand the molecular underpinnings of how these correlated patterns of mRNA expression are generated and maintained.Includes bibliographical reference

Massive transcriptome sequencing of human spinal cord tissues provides new insights into motor neuron degeneration in als

ALS is a devastating and debilitating human disease characterized by the progressive death of upper and lower motor neurons. Although much effort has been made to elucidate molecular determinants underlying the onset and progression of the disorder, the causes of ALS remain largely unknown. In the present work, we have deeply sequenced whole transcriptome from spinal cord ventral horns of post-mortem ALS human donors affected by the sporadic form of the disease (which comprises ∼90% of the cases but which is less investigated than the inherited form of the disease). We observe 1160 deregulated genes including 18 miRNAs and show that down regulated genes are mainly of neuronal derivation while up regulated genes have glial origin and tend to be involved in neuroinflammation or cell death. Remarkably, we find strong deregulation of SNAP25 and STX1B at both mRNA and protein levels suggesting impaired synaptic function through SNAP25 reduction as a possible cause of calcium elevation and glutamate excitotoxicity. We also note aberrant alternative splicing but not disrupted RNA editing

Dynamic Control of Translation During Adult Neurogenesis

Neurons are highly compartmentalized into specific functional units including dendrites, axons and somas. While most messenger RNAs (mRNAs) are constantly used to produce proteins, a subset will remain translationally silent and targeted to aforementioned subcellular structures in order to be available “on demand” upon intra- and extracellular signals. This uncoupling of general availability of mRNAs from actual translation into proteins facilitates immediate response to environmental changes without involving signaling to the soma, which may be far away from axon endings. Furthermore, this way cells avoid excess production of proteins, which is the most energy consuming process within the cell. Adult neural stem cells (NSCs) reside in a thin layer lining the lateral ventricles of the brain and constantly produce progeny that migrates to the olfactory bulb and differentiates into several subtypes of interneurons. Their gene expression has been intensively studied using RNA-based technologies, assuming that mRNA availability readily translates into protein abundance. Whether there is indeed a linear relationship and to which level it is maintained during state transitions throughout neurogenesis has been elusive. Here we studied both global- and transcript-specific translation over multiple stages of neurogenic differentiation. We uncovered dynamic changes of global protein synthesis peaking at stages of proliferation and neuronal integration. Further, using RiboTag mouse models, we showed that transcript abundance and its ribosome-binding shows highest linearity in NSCs that becomes increasingly divergent with the progression of differentiation. NSCs’ transition to early neuroblasts involves translational repression of a subset of mRNAs including both multiple members of the protein synthesis machinery as well as the key pluripotency factor Sox2. In silico motif analysis within this cluster of transcripts led to identification of a pyrimidine-rich motif (PRM) that predicts sensitivity of their translation to the activity of mammalian target of rapamycin complex 1 (mTORC1). Indeed, pharmacological inhibition of mTORC1 reduced ribosome binding of PRM- containing transcripts, while PRM-free transcripts were not affected. Together, this data provides a comprehensive view on the dynamic control of translation during neurogenic differentiation in vivo and uncovers a post-transcriptional mechanism that allows fast and robust repression of pluripotency factors in NSCs as they differentiate