1,721,019 research outputs found
Alternative splice codes for neuronal diversification and synapse specification
Get PDFMammalian nervous systems exhibit an immense structural and functional complexity ranging from billions of neurons to their precise synaptic communication. Neuronal circuits consist of hierarchical assemblies of highly specialized neuronal cell types. Their intrinsic properties and the functional specification of their synapses are fundamental for how circuits process information. However, how diverse classes of neurons establish their cellular and synaptic specificity remains largely unclear. In this thesis, I explored whether cell type-specific alternative splicing programs contribute to the regulation of neuronal and synaptic properties, thereby shaping neuronal connectivity and circuit function.
To investigate whether alternative splicing programs play cell type-specific roles in the mouse brain, I performed global assessments of alternative splicing regulation across neuronal cell classes as well as targeted loss of function studies for one specific alternative splicing regulator. I focused on the RNA binding protein SLM2 which exhibits a remarkable neuronal cell class-specific expression in the mouse brain and had been previously implicated in the regulation of alternative splicing of the synaptic adhesion molecules Neurexin1,2, and 3 (Ehrmann et al., 2013; Iijima et al., 2011). Surprisingly, we found that SLM2 regulates only a handful of transcripts and that loss of SLM2 results in highly selective alterations at glutamatergic synapses in the mouse hippocampus. Genetic correction of the SLM2-dependent target exon of Neurexin 1 was sufficient to rescue synaptic deficits and alterations in the behavior of the Slm2 knock-out animals. Thus, the SLM2 alternative splicing program is highly dedicated to control synapse specification and function in the hippocampus.
In a complementary effort, I investigated how alternative splicing programs are arrayed across different neuronal populations of the forebrain. Systematic mapping of ribosome-associated transcript isoforms in genetically defined cell populations of wild-type animals uncovered extensive transcript isoform diversity across neuronal classes. This revealed that the important drivers for diversification in glutamatergic and GABAergic cells are alternative splicing and transcription start sites. Importantly, we uncovered that such cell class-specific alternative splicing programs mainly target genes implicated in regulating synaptic functions and the intrinsic properties of neurons.
Finally, I explored whether a single RNA binding protein controls common or divergent splicing events and cellular functions in different neuronal populations. We analyzed SLM2-dependent alternative splicing programs in two hippocampal glutamatergic cell classes and somatostatin positive GABAergic neurons. Our findings indicate that there are unique sets of SLM2-dependent transcript isoforms and divergent synaptic phenotypes in different cell populations.
In sum, this work uncovers major roles for cell class-specific alternative splicing programs in the genetic determination of neuronal function and synapse specificatio
Characterization of rostral brainstem nuclei controlling locomotion directionality
No full textRecent studies have uncovered specific functions for brainstem nuclei based on mouse genetics and viral tracing technologies. In this thesis, we characterize pre-motor nuclei of the rostral brainstem and interogate their role in specific full body locomotor functions
Beyond gene expression: post-transcriptional mechanisms for the regulation of neuronal identity and function
Get PDFBrain function relies on complex assemblies of multiple types of excitatory and inhibitory neurons: while the first are responsible for the efficient input processing and transmission, the latter temporally and spatially modulate this flow of information. Each of these neuron types are characterized by distinctive structural, physiological and molecular features, but how this diversity is established during development and maintained throughout adulthood remains one of the most fascinating biomedical problems. Recent studies highlighted how the embryonic differentiation of precursors into specific neuron types is accompanied by finely controlled gene expression signatures. However, the molecular mechanisms that specify properties of mature neurons remain largely unknown.
Neurons exhibit an especially large extent of transcript diversification and regulation by several post-transcriptional mechanisms. In the present work, I investigated whether modulation of RNA processing and metabolism in neurons can define distinct cell types and their unique anatomical and functional properties.
Firstly, in a complementary effort, I performed genetic ribosome tagging in distinct excitatory and inhibitory neuron populations of the mouse brain and performed an extensive genome-wide mapping of ribosome-associated mRNAs. For the first time we identified hundreds of differentially expressed alternative transcripts generated by alternative splicing (AS) and transcription start site usage (ATSS) that can reliably distinguish neuron classes with distinct properties and anatomical localizations in the brain. Interestingly, transcripts that undergo cell type-specific alternative splicing mostly encode proteins critical for synaptic interactions and intrinsic electrical properties of neurons. This demonstrates that AS represents a molecular mechanisms that is particularly tailored to shape and sculpt the characteristic features of functional neurons in a network. Moreover, we further identified differentially expressed RNA-binding proteins that reliably shift splicing patterns of reporters for differentially regulated transcripts. These splicing regulators represent candidates for future in vivo studies on the modulation of respective splicing events.
In a second project, I explored the functional impact of the cell type-selective expression of the RNA-binding protein Rbms3 in GABAergic neurons. In particular, I investigated the molecular mechanism used by Rbms3 to regulate target mRNAs’ metabolism in the cytoplasm of this cell class. Moreover, I showed that genetic ablation of Rbms3 results in both transcriptomic and proteomic defects in the mouse neocortex, which revealed an increase in cellular stress upon Rbms3 loss. Finally, Rbms3 loss-of-function in GABAergic neurons resulted in increased anxiety-related behaviors in mice, suggesting a fundamental role of this RNA-binding protein in modulating the correct functioning of interneurons in the mouse brain.
The miR-17~92 cluster regulates adult neural stem cell behavior
Get PDFIn the adult mammalian brain, the ventricular-subventricular zone (V-SVZ) generates neurons and glia throughout life. In this germinal niche, neural stem cells (NSCs) coexist in quiescent and activated states. However, the molecular mechanisms underlying this transition remain elusive. miRNAs have been implicated in stem cell self-renewal and differentiation, but their role in adult NSC activation is unknown. By performing miRNA profiling of FACS-purified quiescent and activated adult V-SVZ NSCs, we identified the miR-17~92 cluster as highly upregulated in activated stem cells in comparison to their quiescent counterparts. Conditional deletion of miR-17~92 in FACS-purified adult NSCs reduced NSC proliferation in vitro. In vivo, miR-17~92 deletion in NSCs decreased NSC activation, proliferation, and neurogenesis. Unexpectedly, it also led to increased oligodendrogenesis in the V-SVZ, corpus callosum and septum, due to an expansion of OLIG2+ transit-amplifying cells (TACs). Finally, bioinformatic analysis of predicted miR-17~92 targets upregulated in qNSCs versus aNSCs identified S1pr1 and Pdgfrb as promising potential miR-17~92 targets for stem cell activation. In addition, pathway analysis unveiled a gene category related to oligodendrogenesis among the gene categories enriched for miR-17~92 targets. We validated Pdgfrα, a key regulator of oligodendrocyte generation, as a miR-17~92 target by luciferase assay and in vivo analysis. Together, these data uncover multiple functions of the miR-17~92 cluster in adult NSC activation and proliferation, and in the regulation of the balance between neurogenesis and oligodendrogenesis from TACs
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
The role of the Calcium-binding of Copine-6 in synapse function and plasticity
Get PDFThe molecular mechanisms involved in synaptic plasticity are thought to be the basis for the understanding of learning and memory. However, the complexity of the molecular interactions impedes a deep understanding of these mechanisms. Thus far, it has been well established that a common trigger of the synaptic plasticity mechanism is an increase in postsynaptic calcium concentration. Recently, the protein Copine-6 was found as a modulator of synaptic plasticity due to its ability to respond to calcium influx and subsequently to sequester components of the actin cytoskeleton to the postsynaptic membrane of excitatory synapses. Therefore, Copine-6 seems to be a good candidate involved in hippocampal long-term potentiation, learning and memory. Interestingly, Copine-6 has recently been related in different neurological disorders like intellectual disabilities, depression and epilepsy (Anazi et al., 2017; Han et al., 2018; Zhu et al., 2016).
In the last years, our group generated a mouse line in which a calcium-binding mutant of Copine-6 was knocked-into the Cpne6 locus – called Cpne6D167N. Thereafter, we focused on the biochemical characterization of this mouse. We showed that the calcium-dependent enrichment of Copine-6 in membrane fractions of the mouse brain is abrogated in Cpne6D167N mice in the presence of calcium. Importantly, the calcium mutant Copine-6D167N is expressed at the same level as wild-type Copine-6. These data therefore shows that the exchange of Asp to Asn at position 167 of Copine-6 does not affect Copine-6 expression but suppresses its calcium-dependent binding to membranes.
Furthermore, we also demonstrated that calcium binding to Copine-6 is crucial for its ability to act as a synaptic plasticity modulator. We found that expression of Copine-6D167N in the CA1 region of the hippocampus affects the relative proportion of spine types in vivo, as neurons of the hetero- and homozygous knock-in mice express a significantly higher proportion of thin spines at expense of mature spines, a phenotype that was not observed in Cpne6 knock-out (KO) mice. Differences in spine morphology were also observed in primary hippocampal neurons derived from homozygous Cpne6D167N mice, in which an increase in the number of "immature", filopodia-like, thin protrusions and a decrease in mushroom-like protrusions were found. These results suggest that either maturation of spines is delayed or that spines cannot be strengthened following Cpne6D167N mutation. Accordingly, we assessed synaptic strengthening of spines from wild-type, hetero- and homozygous Cpne6D167N neurons by inducing chemical long-term potentiation (cLTP). We found that while wild-type neurons responded with an increased number of mushroom spines and synapses after cLTP induction, phenotypes that have been correlated with synaptic strengthening (Papa et al., 1995; Hosokawa et al., 1995; Fortin et al., 2010) neurons from heterozygous and homozygous Cpne6D167N mice could not respond to the changes related to the cLTP induction paradigm. This suggests that both mutant genotypes failed to undergo synaptic strengthening. Interestingly, heterozygous Cpne6D167N neurons showed elevated numbers of filopodia-like spines after cLTP induction, possibly as a compensatory mechanism to establish synaptic connections. Finally, we also found in Cpne6D167N mice morphological simplifications of CA1 hippocampal pyramidal neurons when compared to wild-type. This result suggests that the binding of calcium to Copine-6 may indirectly affect neuronal morphology as a consequence of spine immaturity.
In conclusion, the calcium-binding site point mutation of Copine-6 seems to have a more profound effect on spine structure plasticity than the complete absence of Copine-6. A similar phenomenon was observed when the phenotypes of mice deficient for CaMKII were compared with mice expressing a phosphorylation mutant of CaMKII (Giese, et al. 1998). Thus, the calcium binding site of Copine-6 seems to be a key element for its ability to act as a calcium sensor and as a further modulator of the synaptic plasticity mechanism. Finally, this work might help to deepen the molecular understanding of synaptic plasticity mechanisms and may also provide new avenues for the molecular understanding of related neurological disorders, revealing possible therapeutic targets
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dealing with aversion: Investigation of the neural substrates for fear and anxiety
Get PDFThe world is a complex and dynamic system, filled with many threats, rewards, and associated cues. To ensure survival, we must deal with incoming threats and cues predictive of threats. Defensive behaviors such as fear and anxiety guide our response to actual and ambiguous threats, respectively. Given their importance to survival, understanding the neural substrates underlying fear and anxiety is paramount but still not complete. With a circuit neuroscience approach, works in my thesis provide evidence that understudied subcortical brain regions such as the lateral Ventral Tegmental Area (VTA) and the Zona Incerta (ZI) contribute to the encoding and modulation of fear and/or anxiety. In the first study, I showed that the lateral VTA gabaergic and glutamatergic neurons encode learned fear. Furthermore, I show that the direct and indirect projections of these lateral VTA subpopulations to the cholinergic interneurons of the dorsal striatum differentially modulate associative fear learning. In my second study, I investigated the ZI and showed that ZI neurons encode cues that contribute to fear learning as well as fear learning itself. In the third and final study, I focused on anxiety and showed that the ZI encode anxiety-related information and actively influence anxiety-like behaviors. Overall, my work advanced our understanding of the neural substrates underlying fear and anxiety
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