RNA-binding proteins (RBPs) regulate genetic diversity, but the degree to which they do so in individual cell types in vivo is unknown. We developed NOVA2 cTag-crosslinking and immunoprecipitation ...(CLIP) to generate functional RBP-RNA maps from different neuronal populations in the mouse brain. Combining cell type datasets from Nova2-cTag and Nova2 conditional knockout mice revealed differential NOVA2 regulatory actions on alternative splicing (AS) on the same transcripts expressed in different neurons. This includes functional differences in transcripts expressed in cortical and cerebellar excitatory versus inhibitory neurons, where we find NOVA2 is required for, respectively, development of laminar structure, motor coordination, and synapse formation. We also find that NOVA2-regulated AS is coupled to NOVA2 regulation of intron retention in hundreds of transcripts, which can sequester the trans-acting splicing factor PTBP2. In summary, cTag-CLIP complements single-cell RNA sequencing (RNA-seq) studies by providing a means for understanding RNA regulation of functional cell diversity.
Display omitted
•cTag-CLIP provides a strategy to study cell-specific RNA regulation in vivo•NOVA2 controls unique RNA splicing programs in inhibitory and excitatory neurons•NOVA2 cTag-CLIP reveals a new mechanism of cell-specific AS regulation•NOVA2 regulates intron retention as a cis-acting scaffold for AS factor PTBP2
cTag-CLIP provides a next-generation strategy to profile functional protein:RNA interactions at cell-specific resolution in vivo. Saito et al. show that differential NOVA2 regulatory actions on alternative splicing coupled to intron removal in different neurons.
Microglia, the macrophages of the brain parenchyma, are key players in neurodegenerative diseases such as Alzheimer's disease. These cells adopt distinct transcriptional subtypes known as states. ...Understanding state function, especially in human microglia, has been elusive owing to a lack of tools to model and manipulate these cells. Here, we developed a platform for modeling human microglia transcriptional states in vitro. We found that exposure of human stem-cell-differentiated microglia to synaptosomes, myelin debris, apoptotic neurons or synthetic amyloid-beta fibrils generated transcriptional diversity that mapped to gene signatures identified in human brain microglia, including disease-associated microglia, a state enriched in neurodegenerative diseases. Using a new lentiviral approach, we demonstrated that the transcription factor MITF drives a disease-associated transcriptional signature and a highly phagocytic state. Together, these tools enable the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.
Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. ...Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3'UTR length. We characterize
, a transcript that shifted from expressing a short 3'UTR isoform to a longer one during granule cell differentiation. We show that
regulates granule cell precursor proliferation and that its long 3'UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies.
Abstract Background Emerging genetic studies of late‐onset Alzheimer’s Disease (LOAD) implicate microglia, the brain’s resident macrophages. More than half the risk genes associated with LOAD are ...expressed in microglia, yet we know little about the underlying biology or how myeloid cells contribute to LOAD pathogenesis. Single‐cell transcriptomic studies reveal diverse microglial states in patients and mouse models; however, we lack tools to track these states and understand the impact of environmental challenges or genetic susceptibility. Method We used human embryonic and induced pluripotent stem cell models to gain insights into microglia. These cells can be differentiated into microglia, enabling the broad characterization of cells in vitro and understanding their impact on the other cell types in xenograft models, allowing us to gain biological insight into disease pathogenesis. Result Our lab has developed a novel human stem cell‐based platform1 which induces stem cell‐derived microglia (iMGLs) to take on diverse transcriptional signatures similar to those found in the human brain in response to exposure to brain‐relevant substrates. Moreover, we conceived a lentiviral protocol that allows for efficient genetic manipulation of microglia (>90% transduction). Building on these systems, we generated reporters of microglia state to track the expression of key microglial genes and adapted iPSC “villages”2, or pooled cultures, that combine iPSC cell lines in the same dish. Using these tools in vitro and in xenograft models3,4, we identified genetic regulators and functional changes of microglia states in LOAD. Conclusion Together, these tools represent a broad toolset that will allow us to answer some of the most pressing questions, such as when and how in disease these states are formed, what is their impact on disease progression, and how plastic they are. These tools will be invaluable to understanding the role of microglia in AD and the brain in general. 1. Dolan, M.‐J. et al. Biorxiv 2022.05.02.490100 (2022) 2. Wells, M. F. et al. Biorxiv 2021.11.08.467815 (2021) 3. Hasselmann, J. et al. Neuron 103 , 1016‐1033.e10 (2019) 4. Mancuso, R. et al. Nature Neuroscience 22 , 2111‐2116 (2019)
Background
Microglia have emerged as key players in the pathogenesis of neurodegenerative conditions such as Alzheimer’s disease (AD), taking on distinct transcriptional and functional states. While ...the ability to profile complex tissues at single‐cell resolution in postmortem brain tissue provides insight into disease‐associated cellular states within the brain, more is required to understand how these changes modulate disease pathogenesis. Building on foundational transcriptomics, the advances in multi‐modal profiling of genomics and proteomics allows for a greater understanding of neuroimmune dysfunction in neurodegenerative disease.
Method
Transcriptomic datasets are often confounded by variability in collection and sequencing methodologies. We have optimized tissue dissociation and cell isolation protocols to avoid many of the pitfalls commonly found in bulk and single‐cell transcriptomic studies. Using these optimized protocols, we have begun assessing paired samples (blood, brain biopsy, and cerebrospinal fluid (CSF), from patients at risk for AD to understand neuroimmune changes in early‐phases of AD. We have also characterized the transcriptional and functional responses of human induced pluripotent stem cell (iPSC) microglia in response to a variety of neurodegenerative brain‐relevant challenges, including amyloid, apoptotic neurons and synaptic debris.
Result
Using scRNA‐seq on fresh mouse and human tissue we have identified a dissociation‐induced signature in microglia that is highly prevalent in current literature, and developed an experimental methodology to prevent this artifact. We have also identified a similar signature in post‐mortem tissue via snRNA‐seq that may be the result of acute‐pre/post‐mortem processes (Marsh et al., 2022). Powerfully, we also have cerebrospinal fluid (CSF) from the same patients, allowing us to correlate proteomic and transcriptomic analyses to determine the connections between disease‐associated transcriptomic cell states and analyte biomarkers. We can then use the iPSC microglia (iMGLs) to understand how altered transcriptomic states and biomarker profiles may alter microglial functions to elucidate mechanisms by which microglia contribute to disease pathogenesis.
Conclusion
Optimization of experimental and analysis methods along with extensive multi‐modal profiling of the same patients will lead to greater understanding of the neuroimmune landscape of neurodegenerative disease to better enable development of novel predictive biomarkers and therapeutic targets.
PDF
