RiboTag: Not Lost in Translation

Lesiak, Atom J.; Neumaier, John F.

doi:10.1038/npp.2015.262

Cited by 7 publications

(6 citation statements)

References 5 publications

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“…One of the obstacles that hamper the investigation is that available microglial cell lines and primary microglia derived from the brain have characteristics different from brain resident microglia because microglia are sensitive to environmental changes. Such obstacles may be circumvented by use of new technologies such as the RiboTag and BacTRAP (Translating Ribosome Affinity Purification) methods (206,207), single-nuclei or single cell RNAseq, genome editing tools, and iPSC-derived 3D co-culture brain models (208). Repeated failures of Aβ-targeted therapeutics indicate the need for a new approach for AD therapy and prevention based on disease mechanisms alternative to the amyloid cascade hypothesis.…”

Section: Discussionmentioning

confidence: 99%

TLR4 Cross-Talk With NLRP3 Inflammasome and Complement Signaling Pathways in Alzheimer's Disease

2020

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Amyloid plaques, mainly composed of abnormally aggregated amyloid β-protein (Aβ) in the brain parenchyma, and neurofibrillary tangles (NFTs), consisting of hyperphosphorylated tau protein aggregates in neurons, are two pathological hallmarks of Alzheimer's disease (AD). Aβ fibrils and tau aggregates in the brain are closely associated with neuroinflammation and synapse loss, characterized by activated microglia and dystrophic neurites. Genome-wide genetic association studies revealed important roles of innate immune cells in the pathogenesis of late-onset AD by recognizing a dozen genetic risk loci that modulate innate immune activities. Furthermore, microglia, brain resident innate immune cells, have been increasingly recognized to play key, opposing roles in AD pathogenesis by either eliminating toxic Aβ aggregates and enhancing neuronal plasticity or producing proinflammatory cytokines, reactive oxygen species, and synaptotoxicity. Aggregated Aβ binds to toll-like receptor 4 (TLR4) and activates microglia, resulting in increased phagocytosis and cytokine production. Complement components are associated with amyloid plaques and NFTs. Aggregated Aβ can activate complement, leading to synapse pruning and loss by microglial phagocytosis. Systemic inflammation can activate microglial TLR4, NLRP3 inflammasome, and complement in the brain, leading to neuroinflammation, Aβ accumulation, synapse loss and neurodegeneration. The host immune response has been shown to function through complex crosstalk between the TLR, complement and inflammasome signaling pathways. Accordingly, targeting the molecular mechanisms underlying the TLR-complement-NLRP3 inflammasome signaling pathways can be a preventive and therapeutic approach for AD.

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Section: Discussionmentioning

confidence: 99%

TLR4 Cross-Talk With NLRP3 Inflammasome and Complement Signaling Pathways in Alzheimer's Disease

2020

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“…RiboTag‐associated RNA extraction as previously described 30,46,47 . The LHb was extracted using a midline 3 mm punch and homogenized in 1 mL of supplemented homogenizing buffer [S‐HB, 50 mM Tris–HCl, 100 mM KCl, 12 mM MgCl2, 1% NP40, 1 mM DTT, 1× Protease inhibitor cocktail (Sigma‐Aldrich), 200 U/mL RNasin (Promega, Madison, WI), 100 μg/mL cyclohexamide (Sigma‐Aldrich), 1 mg/mL heparin (APP Pharmaceuticals, Lake Zurich, IL)].…”

Section: Methodsmentioning

confidence: 99%

“…RiboTag-associated RNA extraction as previously described. 30,46,47 The LHb was extracted using a midline 3 mm punch and homogenized After the final wash, HSB was removed and beads were re-suspended in 400 μL supplemented RLT buffer (10 μL β-mercaptoethanol/10 mL RLT Buffer) from the RNeasy Plus Micro Kit (Qiagen, Hilden, Germany) and vortexed vigorously. These samples were then placed back on the magnet and the RLT buffer was removed from the magnetic beads prior to RNA extraction.…”

Section: Ribotag Extractionmentioning

confidence: 99%

PACAP‐expressing neurons in the lateral habenula diminish negative emotional valence

Levinstein

Bergkamp

Lewis

et al. 2022

Genes Brain and Behavior

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The lateral habenula (LHb) is a small, bilateral, epithalamic nucleus which processes aversive information. While primarily glutamatergic, LHb neurons express genes coding for many neuropeptides, such as Adcyap1 the gene encoding pituitary adenylate cyclase‐activating polypeptide (PACAP), which itself has been associated with anxiety and stress disorders. Using Cre‐dependent viral vectors, we targeted and characterized these neurons based on their anatomical projections and found that they projected to both the raphe and rostromedial tegmentum but only weakly to ventral tegmental area. Using RiboTag to capture ribosomal‐associated mRNA from these neurons and reanalysis of existing single cell RNA sequencing data, we did not identify a unique molecular phenotype that characterized these PACAP‐expressing neurons in LHb. In order to understand the function of these neurons, we conditionally expressed hM3Dq DREADD selectively in LHb PACAP‐expressing neurons and chemogenetically excited these neurons during behavioral testing in the open field test, contextual fear conditioning, sucrose preference, novelty suppressed feeding, and conditioned place preference. We found that Gq activation of these neurons produce behaviors opposite to what is expected from the LHb as a whole—they decreased anxiety‐like and fear behavior and produced a conditioned place preference. In conclusion, PACAP‐expressing neurons in LHb represents a molecularly diverse population of cells that oppose the actions of the remainder of LHb neurons by being rewarding or diminishing the negative consequences of aversive events.

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“…Ultimately, the ability to separate cell-type-specific signatures from these complex populations, by single-cell analyses (Skene et al, 2018;Lang et al, 2019), FACS-based physical separation (Arlotta et al, 2005), biochemical ribosome-tagged separations (Sanz et al, 2009;Lesiak and Neumaier, 2016), and/or computational strategies (for review, see Avila Cobos et al, 2018), will allow the deconvolution of cell-type-specific effects, alone and in coculture with other cell types.…”

Section: Which Cell Types Should Be Studied?mentioning

confidence: 99%

Integrating CRISPR Engineering and hiPSC-Derived 2D Disease Modeling Systems

2020

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Human induced pluripotent stem cells (hiPSCs) have revolutionized research on human diseases, particularly neurodegenerative and psychiatric disorders, making it possible to study mechanisms of disease risk and initiation in otherwise inaccessible patient-specific cells. Today, the integration of CRISPR engineering approaches with hiPSC-based models permits precise isogenic comparisons of human neurons and glia. This review is intended as a guideline for neuroscientists and clinicians interested in translating their research to hiPSC-based studies. It offers state-of-the-art approaches to tackling the challenges that are unique to human in vitro disease models, particularly interdonor and intradonor variability, and limitations in neuronal maturity and circuit complexity. Finally, we provide a detailed overview of the immense possibilities the field has to offer, highlighting efficient neural differentiation and induction strategies for the major brain cell types and providing perspective into integrating CRISPR-based methods into study design. The combination of hiPSC-based disease modeling, CRISPR technology, and high-throughput approaches promises to advance our scientific knowledge and accelerate progress in drug discovery.

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RiboTag: Not Lost in Translation

Cited by 7 publications

References 5 publications

TLR4 Cross-Talk With NLRP3 Inflammasome and Complement Signaling Pathways in Alzheimer's Disease

TLR4 Cross-Talk With NLRP3 Inflammasome and Complement Signaling Pathways in Alzheimer's Disease

PACAP‐expressing neurons in the lateral habenula diminish negative emotional valence

Integrating CRISPR Engineering and hiPSC-Derived 2D Disease Modeling Systems

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