Improved CaP Nanoparticles for Nucleic Acid and Protein Delivery to Neural Primary Cultures and Stem Cells.

Efficiently delivering exogenous materials into primary neurons and neural stem cells (NSCs) has long been a challenge in neurobiology. Existing methods have struggled with complex protocols, unreliable reproducibility, high immunogenicity, and cytotoxicity, causing a huge conundrum and hindering in-depth analyses. Here, we establish a cutting-edge method for transfecting primary neurons and NSCs, named teleofection, by a two-step process to enhance the formation of biocompatible calcium phosphate (CaP) nanoparticles. Teleofection enables both nucleic acid and protein transfection into primary neurons and NSCs, eliminating the need for specialized skills and equipment. It can easily fine-tune transfection efficiency by adjusting the incubation time and nanoparticle quantity, catering to various experimental requirements. Teleofection's versatility allows for the delivery of different cargos into the same cell culture, whether simultaneously or sequentially. This flexibility proves invaluable for long-term studies, enabling the monitoring of neural development and synapse plasticity. Moreover, teleofection ensures the consistent and robust expression of delivered genes, facilitating molecular and biochemical investigations. Teleofection represents a significant advancement in neurobiology, which has promise to transcend the limitations of current gene delivery methods. It offers a user-friendly, cost-effective, and reproducible approach for researchers, potentially revolutionizing our understanding of brain function and development.

CMTR1-Catalyzed 2'-O-Ribose Methylation Controls Neuronal Development by Regulating Camk2α Expression Independent of RIG-I Signaling.

Eukaryotic mRNAs are 5' end capped with a 7-methylguanosine, which is important for processing and translation of mRNAs. Cap methyltransferase 1 (CMTR1) catalyzes 2'-O-ribose methylation of the first transcribed nucleotide (N1 2'-O-Me) to mask mRNAs from innate immune surveillance by retinoic-acid-inducible gene-I (RIG-I). Nevertheless, whether this modification regulates gene expression for neuronal functions remains unexplored. Here, we find that knockdown of CMTR1 impairs dendrite development independent of secretory cytokines and RIG-I signaling. Using transcriptomic analyses, we identify altered gene expression related to dendrite morphogenesis instead of RIG-I-activated interferon signaling, such as decreased calcium/calmodulin-dependent protein kinase 2α (Camk2α). In line with these molecular changes, dendritic complexity in CMTR1-insufficient neurons is rescued by ectopic expression of CaMK2α but not by inactivation of RIG-I signaling. We further generate brain-specific CMTR1-knockout mice to validate these findings in vivo. Our study reveals the indispensable role of CMTR1-catalyzed N1 2'-O-Me in gene regulation for brain development.