CRISPR Based Programmable RNA Editing in Primary Hippocampal Neurons.

Investigating the RNA regulation landscape primarily relies on understanding how RNA-protein interactions are governed in various cell types, including neurons. Analysis of RNA-protein interactions in physiological environments warrants the development of new tools that rely on RNA manipulation. Recently, a CRISPR-based RNA-editing tool (dCas13b-ADAR2DD ) was developed to mitigate disease-associated point mutations in cell lines. Here, we explored the targeted sequence editing potential of the tool (dCas13b-ADAR2DD system) by adapting it to manipulate RNA function to visualize RNA editing in primary hippocampal neurons. This two-component system includes a programmable guide RNA (gRNA) complementary to the target RNA and a catalytically dead version of the Cas13b enzyme fused to ADAR. The RNA editing protocol outlined in this article relies on gRNA-dependent targeting of the dCas13b-ADAR fusion protein to the mutant form of the Dendra2 transcript. Dendra2 is not required for intrinsic cellular functioning. It was ectopically expressed for fluorescent detection as a proof-of-principle demonstration of targeted RNA editing. We first abrogated the fluorescence of Dendra2 by introducing a nonsense mutation that precludes the formation of the functional protein. To visualize the efficacy of the RNA editing in neurons, we used the dCas13b-ADAR2DD system to edit specific nucleotides within the Dendra2 mRNA to restore the amino acid codes critical for Dendra2 fluorescence. This method lays the foundation for future studies on the dynamics of activity-induced RNA-protein interactions in neurons and can be extended to manipulate the endogenous RNome in diverse neuronal subtypes. Furthermore, this methodology will enable investigators to visualize the spatial and temporal resolution of RNA-protein interactions without altering the genomes via conventional methods. © 2023 Wiley Periodicals LLC. Support Protocol: Preparation of mouse primary hippocampal culture Basic Protocol: Targeted editing of RNA.

Defective quality control autophagy in Hyperhomocysteinemia promotes ER stress and consequent neuronal apoptosis through proteotoxicity.

Homocysteine (Hcy), produced physiologically in all cells, is an intermediate metabolite of methionine and cysteine metabolism. Hyperhomocysteinemia (HHcy) resulting from an in-born error of metabolism that leads to accumulation of high levels of Hcy, is associated with vascular damage, neurodegeneration and cognitive decline. Using a HHcy model in neuronal cells, primary cortical neurons and transgenic zebrafish, we demonstrate diminished autophagy and Hcy-induced neurotoxicity associated with mitochondrial dysfunction, fragmentation and apoptosis. We find this mitochondrial dysfunction is due to Hcy-induced proteotoxicity leading to ER stress. We show this sustained proteotoxicity originates from the perturbation of upstream autophagic pathways through an aberrant activation of mTOR and that protetoxic stress act as a feedforward cues to aggravate a sustained ER stress that culminate to mitochondrial apoptosis in HHcy model systems. Using chemical chaperones to mitigate sustained ER stress, Hcy-induced proteotoxicity and consequent neurotoxicity were rescued. We also rescue neuronal lethality by activation of autophagy and thereby reducing proteotoxicity and ER stress. Our findings pave the way to devise new strategies for the treatment of neural and cognitive pathologies reported in HHcy, by either activation of upstream autophagy or by suppression of downstream ER stress. Video Abstract.

Muscleblind-1 interacts with tubulin mRNAs to regulate the microtubule cytoskeleton in C. elegans mechanosensory neurons.

Regulation of the microtubule cytoskeleton is crucial for the development and maintenance of neuronal architecture, and recent studies have highlighted the significance of regulated RNA processing in the establishment and maintenance of neural circuits. In a genetic screen conducted using mechanosensory neurons of C. elegans, we identified a mutation in muscleblind-1/mbl-1 as a suppressor of loss of kinesin-13 family microtubule destabilizing factor klp-7. Muscleblind-1(MBL-1) is an RNA-binding protein that regulates the splicing, localization, and stability of RNA. Our findings demonstrate that mbl-1 is required cell-autonomously for axon growth and proper synapse positioning in the posterior lateral microtubule (PLM) neuron. Loss of mbl-1 leads to increased microtubule dynamics and mixed orientation of microtubules in the anterior neurite of PLM. These defects are also accompanied by abnormal axonal transport of the synaptic protein RAB-3 and reduction of gentle touch sensation in mbl-1 mutant. Our data also revealed that mbl-1 is genetically epistatic to mec-7 (ß tubulin) and mec-12 (α tubulin) in regulating axon growth. Furthermore, mbl-1 is epistatic to sad-1, an ortholog of BRSK/Brain specific-serine/threonine kinase and a known regulator of synaptic machinery, for synapse formation at the correct location of the PLM neurite. Notably, the immunoprecipitation of MBL-1 resulted in the co-purification of mec-7, mec-12, and sad-1 mRNAs, suggesting a direct interaction between MBL-1 and these transcripts. Additionally, mbl-1 mutants exhibited reduced levels and stability of mec-7 and mec-12 transcripts. Our study establishes a previously unknown link between RNA-binding proteins and cytoskeletal machinery, highlighting their crucial roles in the development and maintenance of the nervous system.

Homeostatic scaling is driven by a translation-dependent degradation axis that recruits miRISC remodeling.

Homeostatic scaling in neurons has been attributed to the individual contribution of either translation or degradation; however, there remains limited insight toward understanding how the interplay between the two processes effectuates synaptic homeostasis. Here, we report that a codependence between protein synthesis and degradation mechanisms drives synaptic homeostasis, whereas abrogation of either prevents it. Coordination between the two processes is achieved through the formation of a tripartite complex between translation regulators, the 26S proteasome, and the miRNA-induced silencing complex (miRISC) components such as Argonaute, MOV10, and Trim32 on actively translating transcripts or polysomes. The components of this ternary complex directly interact with each other in an RNA-dependent manner. Disruption of polysomes abolishes this ternary interaction, suggesting that translating RNAs facilitate the combinatorial action of the proteasome and the translational apparatus. We identify that synaptic downscaling involves miRISC remodeling, which entails the mTORC1-dependent translation of Trim32, an E3 ligase, and the subsequent degradation of its target, MOV10 via the phosphorylation of p70 S6 kinase. We find that the E3 ligase Trim32 specifically polyubiquitinates MOV10 for its degradation during synaptic downscaling. MOV10 degradation alone is sufficient to invoke downscaling by enhancing Arc translation through its 3' UTR and causing the subsequent removal of postsynaptic AMPA receptors. Synaptic scaling was occluded when we depleted Trim32 and overexpressed MOV10 in neurons, suggesting that the Trim32-MOV10 axis is necessary for synaptic downscaling. We propose a mechanism that exploits a translation-driven protein degradation paradigm to invoke miRISC remodeling and induce homeostatic scaling during chronic network activity.

Far from the nuclear crowd: Cytoplasmic lncRNA and their implications in synaptic plasticity and memory.

A striking proportion of long non-coding RNAs are expressed specifically in the mammalian brain. Advances in genome-wide sequencing detected widespread diversity in neuronal lncRNAs based on their expression pattern, localization and function. A growing body of literature proposes that localization of lncRNAs is a critical determinant of their function. A rising number of recent findings documented distinct cytoplasmic functions of lncRNAs that are linked to activity-induced control of synaptic plasticity. However, the comprehensive role of cytoplasmic lncRNAs in neuronal functions is less understood. This review surveys our current understanding of lncRNAs that regulate the cytoplasmic life of mRNAs. We discuss the necessity of subcellular localization of lncRNAs in neuronal dendrites and the impact of their compartmentalized positioning on localized translation at the synapse. We have highlighted how lncRNAs modify a functional compartment to meet the demand for input-specific control of synaptic plasticity and memory.

On the functional relevance of spatiotemporally-specific patterns of experience-dependent long noncoding RNA expression in the brain.

The majority of transcriptionally active RNA derived from the mammalian genome does not code for protein. Long noncoding RNA (lncRNA) is the most abundant form of noncoding RNA found in the brain and is involved in many aspects of cellular metabolism. Beyond their fundamental role in the nucleus as decoys for RNA-binding proteins associated with alternative splicing or as guides for the epigenetic regulation of protein-coding gene expression, recent findings indicate that activity-induced lncRNAs also regulate neural plasticity. In this review, we discuss how lncRNAs may exert molecular control over brain function beyond their known roles in the nucleus. We propose that subcellular localization is a critical feature of experience-dependent lncRNA activity in the brain, and that lncRNA-mediated control over RNA metabolism at the synapse serves to regulate local mRNA stability and translation, thereby influencing neuronal function, learning and memory.

Nucleolin regulates 14-3-3ζ mRNA and promotes cofilin phosphorylation to induce tunneling nanotube formation.

Tunneling nanotubes (TNTs) mediate intercellular communication between animal cells in health and disease, but the mechanisms of their biogenesis and function are poorly understood. Here we report that the RNA-binding protein (RBP) nucleolin, which interacts with the known TNT-inducing protein MSec, is essential for TNT formation in mammalian cells. Nucleolin, through its RNA-binding domains (RBDs), binds to and maintains the cytosolic levels of 14-3-3ζ mRNA, and is, therefore, required for TNT formation. A specific region of the 3'-untranslated region (UTR) of the 14-3-3ζ mRNA is likely to be involved in its regulation by nucleolin. Functional complementation experiments suggest that nucleolin and 14-3-3ζ form a linear signaling axis that promotes the phosphorylation and inactivation of the F-actin depolymerization factor cofilin to induce TNT formation. MSec also similarly inactivates cofilin, but potentiates TNT formation independent of the nucleolin-14-3-3ζ axis, despite biochemically interacting with both proteins. We show that 14-3-3ζ and nucleolin are required for the formation of TNTs between primary mouse neurons and astrocytes and in multiple other mammalian cell types. We also report that the Caenorhabditis elegans orthologs of 14-3-3ζ and MSec regulate the size and architecture of the TNT-like cellular protrusions of the distal tip cell (DTC), the germline stem cell niche in the gonad. Our study demonstrates a novel and potentially conserved mRNA-guided mechanism of TNT formation through the maintenance of cellular 14-3-3ζ mRNA levels by the RBP nucleolin.

Reusable glucose sensing using carbon nanotube-based self-assembly.

Lipid functionalized single walled carbon nanotube-based self assembly forms a super-micellar structure. This assemblage has been exploited to trap glucose oxidase in a molecular cargo for glucose sensing. The advantage of such a molecular trap is that all components of this unique structure (both the trapping shell and the entrapped enzyme) are reusable and rechargeable. The unique feature of this sensing method lies in the solid state functionalization of single walled carbon nanotubes that facilitates liquid state immobilization of the enzyme. The method can be used for soft-immobilization (a new paradigm in enzyme immobilization) of enzymes with better thermostability that is imparted by the strong hydrophobic environment provided through encapsulation by the nanotubes.