scholarly journals A Cre-dependent massively parallel reporter assay allows for cell-type specific assessment of the functional effects of genetic variants in vivo

2021 ◽  
Author(s):  
Tomas Lagunas ◽  
Stephen P Plassmyer ◽  
Ryan Z Friedman ◽  
Michael A Rieger ◽  
Anthony D Fischer ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Genetic Variants ◽  
Massively Parallel ◽  
Psychiatric Disease ◽  
Functional Screening ◽  
Cell Type ◽  
Cell Type Specific ◽  
The Impact

Human genetic studies have identified a large number of disease-associated de novo variants in presumptive regulatory regions of the genome that pose a challenge for interpretation of their effects: the impact of regulatory variants is highly dependent on the cellular context, and thus for psychiatric diseases these would ideally be studied in neurons in a living brain. Furthermore, for both common and rare variants, it is expected that only a subset fraction will affect gene expression. Massively Parallel Reporter Assays (MPRAs) are molecular genetic tools that enable functional screening of hundreds of predefined sequences in a single experiment. These assays have been used for functional screening of several different types of regulatory sequences in vitro. However, they have not yet been adapted to query specific cell types in vivo in a complex tissue like the mouse brain. Here, using a test-case 3′UTR MPRA library with variants from ASD patients, we sought to develop a method to achieve reproducible measurements of variant effects in vivo in a cell type-specific manner. We implemented a Cre-dependent design to control expression of our library and first validated our system in vitro. Next, we measured the effect of >500 3′UTR variants in excitatory neurons in the mouse brain. Finally, we report >40 variants with significant effects on transcript abundance in the context of the brain. This new technique should enable robust, functional annotation of genetic variants in the cellular contexts most relevant to psychiatric disease.

scholarly journals Cell type specific profiling of alternative translation identifies novel protein isoforms in the mouse brain

2018 ◽  
Author(s):  
Darshan Sapkota ◽  
Allison M. Lake ◽  
Wei Yang ◽  
Chengran Yang ◽  
Hendrik Wesseling ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Stop Codon ◽  
Affinity Purification ◽  
Protein Isoforms ◽  
Cell Type ◽  
Alternative Translation ◽  
Cell Type Specific ◽  
Ribosome Footprinting

AbstractTranslation canonically begins at a single AUG and terminates at the stop codon, generating one protein species per transcript. However, some transcripts may use alternative initiation sites or sustain translation past their stop codon, generating multiple protein isoforms. Through other mechanisms such as alternative splicing, both neurons and glia exhibit remarkable transcriptional diversity, and these other forms of post-transcriptional regulation are impacted by neural activity and disease. Here, using ribosome footprinting, we demonstrate that alternative translation is likewise abundant in the central nervous system and modulated by stimulation and disease. First, in neuron/glia mixed cultures we identify hundreds of transcripts with alternative initiation sites and confirm the protein isoforms corresponding to a subset of these sites by mass spectrometry. Many of them modulate their alternative initiation in response to KCl stimulation, indicating activity-dependent regulation of this phenomenon. Next, we detect several transcripts undergoing stop codon readthrough thus generating novel C-terminally-extended protein isoforms in vitro. Further, by coupling Translating Ribosome Affinity Purification to ribosome footprinting to enable cell-type specific analysis in vivo, we find that several of both neuronal and astrocytic transcripts undergo readthrough in the mouse brain. Functional analyses of one of these transcripts, Aqp4, reveals readthrough confers perivascular localization, indicating readthrough can be a conserved mechanism to modulate protein function. Finally, we show that AQP4 readthrough is disrupted in multiple gliotic disease models. Our study demonstrates the extensive and regulated use of alternative translational events in the brain and indicates that some of these events alter key protein properties.

scholarly journals Btbd11 is an inhibitory interneuron specific synaptic scaffolding protein that supports excitatory synapse structure and function

2021 ◽  
Author(s):  
Alexei M. Bygrave ◽  
Ayesha Sengupta ◽  
Ella P. Jackert ◽  
Mehroz Ahmed ◽  
Beloved Adenuga ◽  
...  
Keyword(s):  
Inhibitory Interneuron ◽  
Nmda Receptor Antagonist ◽  
Specific Protein ◽  
Network Activity ◽  
Scaffolding Protein ◽  
Inhibitory Interneurons ◽  
Cell Type ◽  
Cell Type Specific

Synapses in the brain exhibit cell–type–specific differences in basal synaptic transmission and plasticity. Here, we evaluated cell–type–specific differences in the composition of glutamatergic synapses, identifying Btbd11, as an inhibitory interneuron–specific synapse–enriched protein. Btbd11 is highly conserved across species and binds to core postsynaptic proteins including Psd–95. Intriguingly, we show that Btbd11 can undergo liquid–liquid phase separation when expressed with Psd–95, supporting the idea that the glutamatergic post synaptic density in synapses in inhibitory and excitatory neurons exist in a phase separated state. Knockout of Btbd11 from inhibitory interneurons decreased glutamatergic signaling onto parvalbumin–positive interneurons. Further, both in vitro and in vivo, we find that Btbd11 knockout disrupts network activity. At the behavioral level, Btbd11 knockout from interneurons sensitizes mice to pharmacologically induced hyperactivity following NMDA receptor antagonist challenge. Our findings identify a cell–type–specific protein that supports glutamatergic synapse function in inhibitory interneurons–with implication for circuit function and animal behavior.

scholarly journals Revisiting the role of IRF3 in inflammation and immunity by conditional and specifically targeted gene ablation in mice

2018 ◽  
Vol 115 (20) ◽  
pp. 5253-5258 ◽  
Author(s):  
Hideyuki Yanai ◽  
Shiho Chiba ◽  
Sho Hangai ◽  
Kohei Kometani ◽  
Asuka Inoue ◽  
...  
Keyword(s):  
Immune Cell ◽  
Cell Types ◽  
Type I ◽  
Type I Ifn ◽  
Cell Type ◽  
Gene Ablation ◽  
Cell Type Specific

IFN regulatory factor 3 (IRF3) is a transcription regulator of cellular responses in many cell types that is known to be essential for innate immunity. To confirm IRF3’s broad role in immunity and to more fully discern its role in various cellular subsets, we engineered Irf3-floxed mice to allow for the cell type-specific ablation of Irf3. Analysis of these mice confirmed the general requirement of IRF3 for the evocation of type I IFN responses in vitro and in vivo. Furthermore, immune cell ontogeny and frequencies of immune cell types were unaffected when Irf3 was selectively inactivated in either T cells or B cells in the mice. Interestingly, in a model of lipopolysaccharide-induced septic shock, selective Irf3 deficiency in myeloid cells led to reduced levels of type I IFN in the sera and increased survival of these mice, indicating the myeloid-specific, pathogenic role of the Toll-like receptor 4–IRF3 type I IFN axis in this model of sepsis. Thus, Irf3-floxed mice can serve as useful tool for further exploring the cell type-specific functions of this transcription factor.

scholarly journals The contribution of astrocyte and neuronal Panx1 to seizures is model and brain region dependent

2021 ◽  
Author(s):  
Price Obot ◽  
Libor Velíšek ◽  
Jana Velíšková ◽  
Eliana Scemes
Keyword(s):  
Brain Region ◽  
Power Spectra ◽  
Cortical Region ◽  
Cell Type ◽  
Epileptiform Discharges ◽  
Seizure Models ◽  
Cell Type Specific ◽  
Specific Deletion

AbstractPannexin1 (Panx1) is an ATP release channel expressed in neurons and astrocytes that plays important roles in CNS physiology and pathology. Evidence for the involvement of Panx1 in seizures includes the reduction of epileptiform activity and ictal discharges following Panx1 channel blockade or deletion. However, very little is known about the relative contribution of astrocyte and neuronal Panx1 channels to hyperexcitability. To this end, mice with global and cell type specific deletion of Panx1 were used in one in vivo and two in vitro seizure models. In the low-Mg2+in vitro model, global deletion but not cell-type specific deletion of Panx1 reduced the frequency of epileptiform discharges. This reduced frequency of discharges did not impact the overall power spectra obtained from local field potentials. In the in vitro KA model, in contrast, global or cell type specific deletion of Panx1 did not affect the frequency of discharges, but reduced the overall power spectra. EEG recordings following KA-injection in vivo revealed that although global deletion of Panx1 did not affect the onset of status epilepticus (SE), SE onset was delayed in mice lacking neuronal Panx1 and accelerated in mice lacking astrocyte Panx1. EEG power spectral analysis disclosed a Panx1-dependent cortical region effect; while in the occipital region, overall spectral power was reduced in all three Panx1 genotypes; in the frontal cortex, the overall power was not affected by deletion of Panx1. Together, our results show that the contribution of Panx1 to ictal activity is model, cell-type and brain region dependent.

scholarly journals The Spectrum of Genetic Variants Associated with the Development of Monogenic Obesity in Qatar

Obesity Facts ◽  
2022 ◽  
Author(s):  
Nadien AbouHashem ◽  
Roan E. Zaied ◽  
Kholoud Al-Shafai ◽  
Mariam Nofal ◽  
Najeeb Syed ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Rare Variants ◽  
Normal Weight ◽  
Routine Testing ◽  
Monogenic Obesity ◽  
Syndromic Obesity ◽  
The Impact

Introduction: Monogenic obesity (MO) is a rare genetic disease characterized by severe early-onset obesity in affected individuals. Previous genetic studies revealed 8 definitive genes for monogenic non-syndromic obesity; many were discovered in consanguineous populations. Here, we examined MO in the Qatari population, whose population is largely consanguineous (54%) and characterized by extensive obesity (45%). Methods: Whole genome sequences of Qatar Biobank samples from 250 subjects with obesity and 250 subjects with normal weight, obtained in association with the Qatar Genome Programme, were searched for genetic variants in the genes known to be associated with MO (i.e., LEP, LEPR, POMC, PCSK1, MC3R, MC4R, MRAP2 and ADCY3). The impact of the variants identified was investigated utilizing in silico tools for prediction in combination with protein visualization by PyMOL. Results: We identified potential MO variants in more than 5% of the cases in our cohort. We revealed 11 rare variants in 6 of the genes targeted, including two disease-causing variants in MC4R and MRAP2, all of which were heterozygous. Moreover, enrichment of a heterozygous ADCY3 variant (c.1658C>T; p.A553V) appeared to cause severe obesity in an autosomal dominant manner. Conclusion: These findings highlight the importance of implementing routine testing for genetic variants that predispose for MO in Qatar. Clearly, additional studies of this nature on populations not yet examined are required. At the same time, functional investigations, both in vitro and in vivo, are necessary in order to better understand the role of the variants identified in the pathogenesis of obesity.

scholarly journals Quantitative secretome analysis establishes the cell type-resolved mouse brain secretome

2020 ◽  
Author(s):  
Johanna Tüshaus ◽  
Stephan A. Müller ◽  
Evans Sioma Kataka ◽  
Jan Zaucha ◽  
Laura Sebastian Monasor ◽  
...  
Keyword(s):  
Mouse Brain ◽  
High Performance ◽  
Brain Slices ◽  
Large Cell ◽  
Cell Type ◽  
Cellular Origin ◽  
Secretome Analysis ◽  
Csf Proteins

AbstractTo understand how cells communicate in the nervous system, it is essential to define their secretome, which is challenging for primary cells because of large cell numbers being required. Here, we miniaturized secretome analysis by developing the high-performance secretome-protein-enrichment-with-click-sugars method (hiSPECS). To demonstrate its broad utility, hiSPECS was used to identify the secretory response of brain slices upon LPS-induced neuroinflammation and to establish the cell type-resolved mouse brain secretome resource using primary astrocytes, microglia, neurons and oligodendrocytes. This resource allowed mapping the cellular origin of CSF proteins and revealed that an unexpectedly high number of secreted proteins in vitro and in vivo are proteolytically-cleaved membrane protein ectodomains. Two examples are neuronally secreted ADAM22 and CD200, which we identified as substrates of the Alzheimer-linked protease BACE1. hiSPECS and the brain secretome resource can be widely exploited to systematically study protein secretion, brain function and to identify cell type-specific biomarkers for CNS diseases.

scholarly journals Cell type-specific biotin labeling in vivo resolves regional neuronal proteomic differences in mouse brain

2021 ◽  
Author(s):  
Sruti Rayaprolu ◽  
Sara Bitarafan ◽  
Ranjita Betarbet ◽  
Sydney N Sunna ◽  
Lihong Cheng ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Neurological Diseases ◽  
Neuronal Cell ◽  
Cell Types ◽  
Proteomic Profiling ◽  
Cell Type ◽  
Specific Expression ◽  
Cell Type Specific ◽  
The Brain

Isolation and proteomic profiling of brain cell types, particularly neurons, pose several technical challenges which limit our ability to resolve distinct cellular phenotypes in neurological diseases. Therefore, we generated a novel mouse line that enables cell type-specific expression of a biotin ligase, TurboID, via Cre-lox strategy for in vivo proximity-dependent biotinylation of proteins. Using adenoviral-based and transgenic approaches, we show striking protein biotinylation in neuronal cell bodies and axons throughout the mouse brain. We quantified more than 2,000 neuron-derived proteins following enrichment that mapped to numerous subcellular compartments. Synaptic, transmembrane transporters, ion channel subunits, and disease-relevant druggable targets were among the most significantly enriched proteins. Remarkably, we resolved brain region-specific proteomic profiles of Camk2a neurons with distinct functional molecular signatures and disease associations that may underlie regional neuronal vulnerability. Leveraging the neuronal specificity of this in vivo biotinylation strategy, we used an antibody-based approach to uncover regionally unique patterns of neuron-derived signaling phospho-proteins and cytokines, particularly in the cortex and cerebellum. Our work provides a proteomic framework to investigate cell type-specific mechanisms driving physiological and pathological states of the brain as well as complex tissues beyond the brain.

scholarly journals DeepG4: A deep learning approach to predict cell-type specific active G-quadruplex regions

2021 ◽  
Vol 17 (8) ◽  
pp. e1009308
Author(s):  
Vincent Rocher ◽  
Matthieu Genais ◽  
Elissar Nassereddine ◽  
Raphael Mourad
Keyword(s):  
Deep Learning ◽  
Double Helix ◽  
Complex Molecule ◽  
Cell Types ◽  
Sequence Motif ◽  
Cell Type ◽  
G Quadruplex ◽  
Cell Type Specific

DNA is a complex molecule carrying the instructions an organism needs to develop, live and reproduce. In 1953, Watson and Crick discovered that DNA is composed of two chains forming a double-helix. Later on, other structures of DNA were discovered and shown to play important roles in the cell, in particular G-quadruplex (G4). Following genome sequencing, several bioinformatic algorithms were developed to map G4s in vitro based on a canonical sequence motif, G-richness and G-skewness or alternatively sequence features including k-mers, and more recently machine/deep learning. Recently, new sequencing techniques were developed to map G4s in vitro (G4-seq) and G4s in vivo (G4 ChIP-seq) at few hundred base resolution. Here, we propose a novel convolutional neural network (DeepG4) to map cell-type specific active G4 regions (e.g. regions within which G4s form both in vitro and in vivo). DeepG4 is very accurate to predict active G4 regions in different cell types. Moreover, DeepG4 identifies key DNA motifs that are predictive of G4 region activity. We found that such motifs do not follow a very flexible sequence pattern as current algorithms seek for. Instead, active G4 regions are determined by numerous specific motifs. Moreover, among those motifs, we identified known transcription factors (TFs) which could play important roles in G4 activity by contributing either directly to G4 structures themselves or indirectly by participating in G4 formation in the vicinity. In addition, we used DeepG4 to predict active G4 regions in a large number of tissues and cancers, thereby providing a comprehensive resource for researchers. Availability: https://github.com/morphos30/DeepG4.

scholarly journals Induction of Golli-MBP Expression in CNS Macrophages During Acute LPS-Induced CNS Inflammation and Experimental Autoimmune Encephalomyelitis (EAE)

2007 ◽  
Vol 7 ◽  
pp. 112-120 ◽  
Author(s):  
Tracey L. Papenfuss ◽  
J. Cameron Thrash ◽  
Patricia E. Danielson ◽  
Pamela E. Foye ◽  
Brian S. Hllbrush ◽  
...  
Keyword(s):  
Experimental Autoimmune Encephalomyelitis ◽  
Cell Type ◽  
Autoimmune Encephalomyelitis ◽  
Cns Inflammation ◽  
Cell Type Specific ◽  
Candidate Molecule ◽  
Experimental Autoimmune

Microglia are the tissue macrophages of the CNS. Microglial activation coupled with macrophage infiltration is a common feature of many classic neurodegenerative disorders. The absence of cell-type specific markers has confounded and complicated the analysis of cell-type specific contributions toward the onset, progression, and remission of neurodegeneration. Molecular screens comparing gene expression in cultured microglia and macrophages identified Golli-myelin basic protein (MBP) as a candidate molecule enriched in peripheral macrophages.In situhybridization analysis of LPS/IFNg and experimental autoimmune encephalomyelitis (EAE)–induced CNS inflammation revealed that only a subset of CNS macrophages express Golli-MBP. Interestingly, the location and morphology of Golli-MBP+ CNS macrophages differs between these two models of CNS inflammation. These data demonstrate the difficulties of extendingin vitroobservations toin vivobiology and concretely illustrate the complex heterogeneity of macrophage activation states present in region- and stage-specific phases of CNS inflammation. Taken altogether, these are consistent with the emerging picture that the phenotype of CNS macrophages is actively defined by their molecular interactions with the CNS microenvironment.

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