Project description:<p>The goal of this proposal is to bring together the power of 1) whole exome sequencing, 2) homozygosity mapping in consanguineous families, 3) genome-wide maps of neuronal transcription in response to neuronal activity, and 4) genome-wide maps of the binding sites of factors that regulate this transcription to generate and annotate a catalog of ASD-associated variants. The consanguineous families are already enrolled in research, and have been phenotyped. The neuronal transcription and binding site maps will be developed by the Greenberg Lab at Harvard Medical School. The whole exome sequencing will be done at the Broad Institute, and the Walsh lab at Children's Hospital will validate the results and analyze the variant data.</p>
Project description:Inhibition of Brd4 with Jq1 in neurons with or without BDNF stimulation Examination of the effects of Jq1 treatment on primary mouse cortical neurons
Project description:Maintaining an appropriate balance between excitation and inhibition is critical for information processing in cortical neurons. It is known that cortical neurons receive widely disparate levels of excitation. To ensure efficient coding, they are capable of cell-autonomously adjusting the inhibition they receive to the individual levels of excitatory input, but the underlying mechanisms are not understood.
The article associated with this data shows that Ste20-like kinase (SLK) in cortical neurons mediates cell-autonomous regulation of excitation-inhibition balance in the thalamocortical feed-forward circuit, but not in the feed-back circuit. The parallel reaction monitoring data here supports the link between activity, SLK phosphorylation and function.
Project description:Protein coding gene expression requires two steps – transcription and translation – which can be regulated independently to allow nuanced, localized, and rapid responses to cellular stimuli. Neurons are known to respond transcriptionally and translationally to bursts of brain activity, and a transcriptional response to this activation has also been recently characterized in astrocytes. However, the extent to which astrocytes respond translationally is unknown. We tested the hypothesis that astrocytes also have a programmed translational response by characterizing the change in transcript ribosome occupancy in astrocytes using Translating Ribosome Affinity Purification subsequent to a robust induction of neuronal activity in vivo via acute seizure. We identified a reproducible change in transcripts on astrocyte ribosomes, highlighted by a rapid decrease in housekeeping transcripts, such as ribosomal and mitochondrial components, and a rapid increase in transcripts related to cytoskeleton, motor activity, ion transport, and cell communication. This indicates a dynamic response, some of which might be secondary to activation of Receptor Tyrosine Kinase signaling. Using acute slices, we quantified the extent to which individual cues and sequela of neuronal activity can activate translation acutely in astrocytes. This identified both BDNF and KCl as contributors to translation induction, the latter with both action-potential sensitive and insensitive components. Finally, we show that this translational response requires the presence of neurons, indicating the response is acutely or chronically non-cell autonomous. Regulation of translation in astrocytes by neuronal activity suggests an additional mechanism by which astrocytes may dynamically modulate nervous system functioning.
Project description:HEK-293 cells transfected with non-targeting control siRNA or UPF1LL-specific siRNA were treated with vehicle control, puromycin, or thapsigargin as indicated and used for total RNA-seq.
Project description:miRNAs play important roles in every aspect of plant development. Small RNA regulation confers sensitivity and robustness onto gene regulatory networks, and the morphogen-like readout of small RNA mobility gradients yields sharply delineated domains of target gene expression. However, how the spatiotemporal patterns of miRNA activity are attained is less well understood. We used the high-resolution maize shoot apex transcriptome atlas (Knauer et al., 2019) together with data from small RNA-Seq, PARE analysis, small RNA in-situ hybridization, and RNAseq of weak dcl1 mutant tissues to demonstrate that miRNA accumulation and function during development is regulated in a tiissue dependent manner at the transcriptional and post-transcriptional level.