Subcellular sequencing of single neurons reveals the dendritic transcriptome of GABAergic interneurons

Although mRNAs are localized in the processes of excitatory neurons, it is still unclear whether interneurons also localize a large population of mRNAs. In addition, the variability in the localized mRNA population within and between cell-types is unknown. Here we describe the unbiased transcriptomic characterization of the subcellular compartments of hundreds of single neurons. We separately profiled the dendritic and somatic transcriptomes of individual rat hippocampal neurons and investigated mRNA abundances in the soma and dendrites of single glutamatergic and GABAergic neurons. We found that, like their excitatory counterparts, interneurons contain a rich repertoire of ~4000 mRNAs. We observed more cell type-specific features among somatic transcriptomes than their associated dendritic transcriptomes. Finally, using cell-type specific metabolic labelling of isolated neurites, we demonstrated that the processes of Glutamatergic and, notably, GABAergic neurons were capable of local translation, suggesting mRNA localization and local translation is a general property of neurons

The translatome of neuronal cell bodies, dendrites,and axons

To form synaptic connections and store information, neurons continuously remodel their proteomes. The impressive length of dendrites and axons imposes logistical challenges to maintain synaptic proteins at locations remote from the transcription source (the nucleus). The discovery of thousands of messenger RNAs (mRNAs) near synapses suggested that neurons overcome distance and gain autonomy by producing proteins locally. It is not generally known, however, if, how, and when localized mRNAs are translated into protein. To investigate the translational landscape in neuronal subregions, we performed simultaneous RNA sequencing (RNA-seq) and ribosome sequencing (Ribo-seq) from microdissected rodent brain slices to identify and quantify the transcriptome and translatome in cell bodies (somata) as well as dendrites and axons (neuropil). Thousands of transcripts were differentially translated between somatic and synaptic regions, with many scaffold and signaling molecules displaying increased translation levels in the neuropil. Most translational changes between compartments could be accounted for by differences in RNA abundance. Pervasive translational regulation was observed in both somata and neuropil influenced by specific mRNA features (e.g., untranslated region [UTR] length, RNA-binding protein [RBP] motifs, and upstream open reading frames [uORFs]). For over 800 mRNAs, the dominant source of translation was the neuropil. We constructed a searchable and interactive database for exploring mRNA transcripts and their translation levels in the somata and neuropil [MPI Brain Research, The mRNA translation landscape in the synaptic neuropil. https://public.brain.mpg.de/dashapps/localseq/ Accessed 5 October 2021]. Overall, our findings emphasize the substantial contribution of local translation to maintaining synaptic protein levels and indicate that on-site translational control is an important mechanism to control synaptic strength

Full-length transcriptome reconstruction reveals a large diversity of RNA and protein isoforms in rat hippocampus

Gene annotation is a critical resource in genomics research. Many computational approaches have been developed to assemble transcriptomes based on high-throughput short-read sequencing, however, only with limited accuracy. Here, we combine next-generation and third-generation sequencing to reconstruct a full-length transcriptome in the rat hippocampus, which is further validated using independent 5´ and 3´-end profiling approaches. In total, we detect 28,268 full-length transcripts (FLTs), covering 6,380 RefSeq genes and 849 unannotated loci. Based on these FLTs, we discover co-occurring alternative RNA processing events. Integrating with polysome profiling and ribosome footprinting data, we predict isoform-specific translational status and reconstruct an open reading frame (ORF)-eome. Notably, a high proportion of the predicted ORFs are validated by mass spectrometry-based proteomics. Moreover, we identify isoforms with subcellular localization pattern in neurons. Collectively, our data advance our knowledge of RNA and protein isoform diversity in the rat brain and provide a rich resource for functional studies

Advancing brain barriers RNA sequencing: guidelines from experimental design to publication

Background: RNA sequencing (RNA-Seq) in its varied forms has become an indispensable tool for analyzing differential gene expression and thus characterization of specific tissues. Aiming to understand the brain barriers genetic signature, RNA seq has also been introduced in brain barriers research. This has led to availability of both, bulk and single-cell RNA-Seq datasets over the last few years. If appropriately performed, the RNA-Seq studies provide powerful datasets that allow for significant deepening of knowledge on the molecular mechanisms that establish the brain barriers. However, RNA-Seq studies comprise complex workflows that require to consider many options and variables before, during and after the proper sequencing process.Main body: In the current manuscript, we build on the interdisciplinary experience of the European PhD Training Network BtRAIN (https://www.btrain-2020.eu/) where bioinformaticians and brain barriers researchers collaborated to analyze and establish RNA-Seq datasets on vertebrate brain barriers. The obstacles BtRAIN has identified in this process have been integrated into the present manuscript. It provides guidelines along the entire workflow of brain barriers RNA-Seq studies starting from the overall experimental design to interpretation of results. Focusing on the vertebrate endothelial blood–brain barrier (BBB) and epithelial blood-cerebrospinal-fluid barrier (BCSFB) of the choroid plexus, we provide a step-by-step description of the workflow, highlighting the decisions to be made at each step of the workflow and explaining the strengths and weaknesses of individual choices made. Finally, we propose recommendations for accurate data interpretation and on the information to be included into a publication to ensure appropriate accessibility of the data and reproducibility of the observations by the scientific community.Conclusion: Next generation transcriptomic profiling of the brain barriers provides a novel resource for understanding the development, function and pathology of these barrier cells, which is essential for understanding CNS homeostasis and disease. Continuous advancement and sophistication of RNA-Seq will require interdisciplinary approaches between brain barrier researchers and bioinformaticians as successfully performed in BtRAIN. The present guidelines are built on the BtRAIN interdisciplinary experience and aim to facilitate collaboration of brain barriers researchers with bioinformaticians to advance RNA-Seq study design in the brain barriers community

Evolution of pallium, hippocampus and cortical cell types revealed by single-cell transcriptomics in reptiles

Computations in the mammalian cortex are carried out by glutamatergic and GABAergic neurons forming specialized circuits and areas. Here we asked how these neurons and areas evolved in amniotes. We built a gene expression altas of the pallium of two reptilian species using large-scale single-cell mRNA sequencing. The transcriptomic signature of glutamatergic neurons in reptilian cortex suggests that mammalian neocortical layers are made of new cell types generated by diversification of ancestral gene regulatory programs. By contrast, the diversity of reptilian cortical GABAergic neurons indicates that the interneuron classes known in mammals already existed in the common ancestor of all amniotes

Alternative 3' UTRs Modify the Localization, Regulatory Potential, Stability, and Plasticity of mRNAs in Neuronal Compartments

Neurons localize mRNAs near synapses where their translation can be regulated by synaptic demand and activity. Differences in the 3' UTRs of mRNAs can change their localization, stability, and translational regulation. Using 3' end RNA sequencing of microdissected rat brain slices, we discovered a huge diversity in mRNA 3' UTRs, with many transcripts showing enrichment for a particular 3' UTR isoform in either somata or the neuropil. The 3' UTR isoforms of localized transcripts are significantly longer than the 3' UTRs of non-localized transcripts and often code for proteins associated with axons, dendrites, and synapses. Surprisingly, long 3' UTRs add not only new, but also duplicate regulatory elements. The neuropil-enriched 3' UTR isoforms have significantly longer half-lives than somata-enriched isoforms. Finally, the 3' UTR isoforms can be significantly altered by enhanced activity. Most of the 3' UTR plasticity is transcription dependent, but intriguing examples of changes that are consistent with altered stability, trafficking between compartments, or local "remodeling" remain

EXPERIENCE WITH SURGICAL TREATMENT IN PATIENTS WITH LYMPHOPROLIFERATIVE LESION IN THE BRAIN AND SKULL VAULT BONES

The problem of lymphoproliferative lesions of the central nervous system (CNS) is urgent in both patients with immunodeficiency states and those without background systemic pathology in spite of the fact that the statistics vary greatly in these two categories. Despite a wide range of noninvasive diagnostic procedures, it is impossible to obtain a significant confirmation of primary lymphoproliferative CNS lesion in patients with no background systemic manifestations of the disease. Stereotactic biopsy, followed by combination chemo- and radiotherapy in accordance with the existing protocols, is a well-known procedure to treat suspected primary CNS lymphoma. This paper describes 4 cases of primary CNS and skull bone lymphomas in patients operated on for skull bone granuloma (in one case) or rapid progression of focal and global cerebral neurological deficit (in 3 cases). A postoperative histological analysis using the currently available methods revealed lymphoproliferative lesions; in this connection the patients continued treatment by oncologists

Evolution of pallium, hippocampus, and cortical cell types revealed by single-cell transcriptomics in reptiles

Computations in the mammalian cortex are carried out by glutamatergic and γ-aminobutyric acid–releasing (GABAergic) neurons forming specialized circuits and areas. Here we asked how these neurons and areas evolved in amniotes. We built a gene expression atlas of the pallium of two reptilian species using large-scale single-cell messenger RNA sequencing. The transcriptomic signature of glutamatergic neurons in reptilian cortex suggests that mammalian neocortical layers are made of new cell types generated by diversification of ancestral gene-regulatory programs. By contrast, the diversity of reptilian cortical GABAergic neurons indicates that the interneuron classes known in mammals already existed in the common ancestor of all amniotes

Alternative polyadenylation and differential expression of Shank mRNAs in the synaptic neuropil

The stability and dynamics of synapses rely on tight regulation of the synaptic proteome. Shank proteins, encoded by the three genes Shank1, Shank2 and Shank3 are scaffold molecules in the postsynaptic density of excitatory neurons that contribute to activity-dependent neuronal signalling. Mutations in the Shank genes are associated with neurological diseases. Using state-of-the-art technologies, we investigated the levels of expression of the Shank family messenger RNAs (mRNAs) within the synaptic neuropil of the rat hippocampus. We detected all three Shank transcripts in the neuropil of CA1 pyramidal neurons. We found Shank1 to be the most abundantly expressed among the three Shank mRNA homologues. We also examined the turnover of Shank mRNAs and predict the half-lives of Shank1, Shank2 and Shank3 mRNAs to be 18-28 h. Using 3'-end sequencing, we identified novel 3' ends for the Shank1 and Shank2 3' untranslated regions (3' UTRs) that may contribute to the diversity of alternative polyadenylation (APA) for the Shank transcripts. Our findings consolidate the view that the Shank molecules play a central role at the postsynaptic density. This study may shed light on synaptopathologies associated with disruption of local protein synthesis, perhaps linked to mutations in mRNA 3' UTRs or inappropriate 3' end processing