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.

Dysregulated proteostasis network in neuronal diseases.

Long-term maintenance of synaptic connections is important for brain function, which depends on varying proteostatic regulations to govern the functional integrity of neuronal proteomes. Proteostasis supports an interconnection of pathways that regulates the fate of proteins from synthesis to degradation. Defects in proteostatic signaling are associated with age-related functional decline and neurodegenerative diseases. Recent studies have advanced our knowledge of how cells have evolved distinct mechanisms to safely control protein homeostasis during synthesis, folding and degradation, and in different subcellular organelles and compartments. Neurodegeneration occurs when these protein quality controls are compromised by accumulated pathogenic proteins or aging to an irreversible state. Consequently, several therapeutic strategies, such as targeting the unfolded protein response and autophagy pathways, have been developed to reduce the burden of misfolded proteins and proved useful in animal models. Here, we present a brief overview of the molecular mechanisms involved in maintaining proteostatic networks, along with some examples linking dysregulated proteostasis to neuronal diseases.

Endogenous Expression of G-CSF in Rat Dorsal Root Ganglion Neurons after Nerve Injury.

Granulocyte colony-stimulating factor (G-CSF) has been reported to modulate pain function following nerve injury. However, the expression of endogenous G-CSF in the dorsal root ganglion (DRG) and the response to nerve injury remain unclear. In the present study, we demonstrated that G-CSF and G-CSFR are mainly expressed in both small- and medium-diameter DRG neurons in rats and are responsible for transmitting pain responses. G-CSF and G-CSFR were co-expressed in certain nociceptive DRG neurons. In addition, G-CSF was expressed in satellite glial cells around large-diameter DRG neurons. After sciatic nerve injury, the number of G-CSF-positive DRG neurons was increased in both the ipsilateral and contralateral lesion sites in rats. However, G-CSF expression in satellite glial cells was not affected by nerve injury. To clarify the role of G-CSF in pain, exogenous G-CSF was administered to a rat model of neuropathic pain induced by partial sciatic nerve transaction (PST). Our results indicate that treatment with G-CSF did not attenuate but exacerbated neuropathic pain. In summary, G-CSF may directly activate sensory neurons and contribute to nociceptive signaling.

COUP-TFI specifies the medial entorhinal cortex identity and induces differential cell adhesion to determine the integrity of its boundary with neocortex.

Development of cortical regions with precise, sharp, and regular boundaries is essential for physiological function. However, little is known of the mechanisms ensuring these features. Here, we show that determination of the boundary between neocortex and medial entorhinal cortex (MEC), two abutting cortical regions generated from the same progenitor lineage, relies on COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I), a patterning transcription factor with graded expression in cortical progenitors. In contrast with the classical paradigm, we found that increased COUP-TFI expression expands MEC, creating protrusions and disconnected ectopic tissue. We further developed a mathematical model that predicts that neuronal specification and differential cell affinity contribute to the emergence of an instability region and boundary sharpness. Correspondingly, we demonstrated that high expression of COUP-TFI induces MEC cell fate and protocadherin 19 expression. Thus, we conclude that a sharp boundary requires a subtle interplay between patterning transcription factors and differential cell affinity.

CPEB4-Dependent Neonate-Born Granule Cells Are Required for Olfactory Discrimination.

The rodent olfactory bulb (OB) contains two distinct populations of postnatally born interneurons, mainly granule cells (GCs), to support local circuits throughout life. During the early postnatal period (i.e., 2 weeks after birth), GCs are mostly produced locally from progenitor cells in the OB with a proportion of them deriving from proliferating cells in the rostral migratory stream (RMS). Afterward, the replenishment of GCs involves differentiated neuroblasts from the subventricular zone (SVZ) in a process known as adult neurogenesis. Although numerous studies have addressed the role of SVZ-born GCs in olfactory behaviors, the function of GCs produced early postnatally in the OB remains elusive. Our previous study demonstrated that the translational regulator, cytoplasmic polyadenylation element-binding protein 4 (CPEB4), is a survival factor exclusively for neonate-born but not SVZ/adult-derived GCs, so CPEB4-knockout (KO) mice provide unique leverage to study early postnatal-born GC-regulated olfactory functions. CPEB4-KO mice with hypoplastic OBs showed normal olfactory sensitivity and short-term memory, but impaired ability to spontaneously discriminate two odors. Such olfactory dysfunction was recapitulated in specific ablation of Cpeb4 gene in inhibitory interneurons but not in excitatory projection neurons or SVZ-derived interneurons. The continuous supply of GCs from adult neurogenesis eventually restored the OB size but not the discrimination function in 6-month-old KO mice. Hence, in the early postnatal OB, whose function cannot be replaced by adult-born GCs, construct critical circuits for odor discrimination.

RBPMetaDB: a comprehensive annotation of mouse RNA-Seq datasets with perturbations of RNA-binding proteins.

RNA-binding proteins (RBPs) may play a critical role in gene regulation in various diseases or biological processes by controlling post-transcriptional events such as polyadenylation, splicing and mRNA stabilization via binding activities to RNA molecules. Owing to the importance of RBPs in gene regulation, a great number of studies have been conducted, resulting in a large amount of RNA-Seq datasets. However, these datasets usually do not have structured organization of metadata, which limits their potentially wide use. To bridge this gap, the metadata of a comprehensive set of publicly available mouse RNA-Seq datasets with perturbed RBPs were collected and integrated into a database called RBPMetaDB. This database contains 292 mouse RNA-Seq datasets for a comprehensive list of 187 RBPs. These RBPs account for only ∼10% of all known RBPs annotated in Gene Ontology, indicating that most are still unexplored using high-throughput sequencing. This negative information provides a great pool of candidate RBPs for biologists to conduct future experimental studies. In addition, we found that DNA-binding activities are significantly enriched among RBPs in RBPMetaDB, suggesting that prior studies of these DNA- and RNA-binding factors focus more on DNA-binding activities instead of RNA-binding activities. This result reveals the opportunity to efficiently reuse these data for investigation of the roles of their RNA-binding activities. A web application has also been implemented to enable easy access and wide use of RBPMetaDB. It is expected that RBPMetaDB will be a great resource for improving understanding of the biological roles of RBPs.Database URL: http://rbpmetadb.yubiolab.org.

SFMetaDB: a comprehensive annotation of mouse RNA splicing factor RNA-Seq datasets.

Although the number of RNA-Seq datasets deposited publicly has increased over the past few years, incomplete annotation of the associated metadata limits their potential use. Because of the importance of RNA splicing in diseases and biological processes, we constructed a database called SFMetaDB by curating datasets related with RNA splicing factors. Our effort focused on the RNA-Seq datasets in which splicing factors were knocked-down, knocked-out or over-expressed, leading to 75 datasets corresponding to 56 splicing factors. These datasets can be used in differential alternative splicing analysis for the identification of the potential targets of these splicing factors and other functional studies. Surprisingly, only ∼15% of all the splicing factors have been studied by loss- or gain-of-function experiments using RNA-Seq. In particular, splicing factors with domains from a few dominant Pfam domain families have not been studied. This suggests a significant gap that needs to be addressed to fully elucidate the splicing regulatory landscape. Indeed, there are already mouse models available for ∼20 of the unstudied splicing factors, and it can be a fruitful research direction to study these splicing factors in vitro and in vivo using RNA-Seq. Database URL:http://sfmetadb.ece.tamu.edu/

Olfactory-Experience- and Developmental-Stage-Dependent Control of CPEB4 Regulates c-Fos mRNA Translation for Granule Cell Survival.

Mammalian olfactory bulbs (OBs) require continuous replenishment of interneurons (mainly granule cells [GCs]) to support local circuits throughout life. Two spatiotemporally distinct waves of postnatal neurogenesis contribute to expanding and maintaining the GC pool. Although neonate-born GCs have a higher survival rate than adult-born GCs, the molecular mechanism underlying this survival remains unclear. Here, we find that cytoplasmic polyadenylation element-binding protein 4 (CPEB4) acts as a survival factor exclusively for early postnatal GCs. In mice, during the first 2 postnatal weeks, olfactory experience initiated CPEB4-activated c-Fos mRNA translation. In CPEB4-knockout mice, c-FOS insufficiency reduced neurotrophic signaling to impair GC survival and cause OB hypoplasia. Both cyclic AMP responsive element binding protein (CREB)-dependent transcription and CPEB4-promoted translation support c-FOS expression early postnatal OBs but disengage in adult OBs. Activity-related c-FOS synthesis and GC survival are thus developmentally controlled by distinct molecular mechanisms to govern OB growth.