Astroglia play active and diverse roles in modulating neuronal/synaptic functions in the CNS. How these astroglial functions are regulated, especially by neuronal signals, remains largely unknown. Exosomes, a major type of extracellular vesicles (EVs) that originate from endosomal intraluminal vesicles (ILVs), have emerged as a new intercellular communication process. By generating cell-type-specific ILVs/exosome reporter (CD63-GFPf/f) mice and immuno-EM/confocal image analysis, we found that neuronal CD63-GFP+ ILVs are primarily localized in soma and dendrites, but not in axonal terminals in vitro and in vivo. Secreted neuronal exosomes contain a subset of microRNAs (miRs) that is distinct from the miR profile of neurons. These miRs, especially the neuron-specific miR-124-3p, are potentially internalized into astrocytes. MiR-124-3p further up-regulates the predominant glutamate transporter GLT1 by suppressing GLT1-inhibiting miRs. Our findings suggest a previously undescribed neuronal exosomal miR-mediated genetic regulation of astrocyte functions, potentially opening a new frontier in understanding CNS intercellular communication.

Developing safe and efficient delivery systems for therapeutic biomacromolecules is a long‐standing challenge. Herein, we report a newly developed combinatorial library of cholesteryl‐based disulfide bond‐containing biodegradable cationic lipidoid nanoparticles. We have identified a subset of this library which is effective for protein and mRNA delivery in vitro and in vivo. These lipidoids showed comparable transfection efficacies but much lower cytotoxicities compared to the Lpf2k in vitro. In vivo studies in adult mice demonstrated the successful delivery of genome engineering protein and mRNA molecules in the skeletal muscle (via intramuscular injection), lung and spleen (via intravenous injection), and brain (via lateral ventricle infusion).

Alteration of glutamatergic synaptic plasticity in the Nucleus Accumbens (NAc) has been implicated in cocaine-seeking behaviors. Astroglial mechanisms for maintaining extracellular glutamate homeostasis through cysteine/glutamate exchanger (xCT) and glutamate transporter GLT1 are dysregulated following cocaine exposure and contribute to altered glutamatergic synaptic plasticity. However, how these astroglial proteins become dysregulated in cocaine addiction remains unknown. We recently showed that neuron to astroglial exosome signaling is essential to maintain GLT1 protein expression by transferring neuronal miR-124-3p into astrocytes to suppress GLT1-inhibiting microRNAs (miRs) in astrocytes. In the current study, by selectively labeling neuronal exosomes using CD63-GFP f/+ exosome reporter mice, we examined how the self-administration and extinction stages of the mouse cocaine self-administration model alter neuronal exosome signaling to astrocytes and microglia in the NAc. We found that cocaine (but not food) self-administration strongly reduces the internalization of neuronal exosomes, particularly in astrocytes in the NAc (but not in motor cortex), which can be effectively reversed by extinction training. In parallel, cocaine self-administration alone specifically and differentially affects activation of glial cells by decreasing GFAP expression in astrocytes but increasing Iba1 expression in microglia. However, extinction training fully reverses the increased Iba1 expression in microglia but only partially reverses the reduction of GFAP in astrocytes. Taken together, our study reveals altered in vivo dynamics of NAc neuronal exosomes in the cocaine addiction model, providing new insights about how altered neuron to glial exosome signaling may contribute to astroglial dysfunction in cocaine addiction.

Developing safe and efficient delivery systems for therapeutic biomacromolecules is a long‐standing challenge. Herein, we report a newly developed combinatorial library of cholesteryl‐based disulfide bond‐containing biodegradable cationic lipidoid nanoparticles. We have identified a subset of this library which is effective for protein and mRNA delivery in vitro and in vivo. These lipidoids showed comparable transfection efficacies but much lower cytotoxicities compared to the Lpf2k in vitro. In vivo studies in adult mice demonstrated the successful delivery of genome engineering protein and mRNA molecules in the skeletal muscle (via intramuscular injection), lung and spleen (via intravenous injection), and brain (via lateral ventricle infusion).

Motor neurons (MNs) innervating the digit muscles of the intrinsic hand (IH) and intrinsic foot (IF) control fine motor movements. The ability to reproducibly label specifically IH and IF MNs in mice would be a beneficial tool for studies focused on fine motor control. To this end, we find that a CRE knock-in mouse line of Atoh1 , a developmentally expressed basic helix-loop-helix (bHLH) transcription factor, reliably expresses CRE-dependent reporter genes in ∼60% of the IH and IF MNs. We determine that CRE-dependent expression in IH and IF MNs is ectopic because an Atoh1 mouse line driving FLPo recombinase does not label these MNs although other Atoh1 -lineage neurons in the intermediate spinal cord are reliably identified. Furthermore, the CRE-dependent reporter expression is enriched in the IH and IF MN pools with much sparser labeling of other limb-innervating MN pools such as the tibialis anterior (TA), gastrocnemius (GS), quadricep (Q), and adductor (Ad). Lastly, we find that ectopic reporter expression begins postnatally and labels a mixture of α and γ-MNs. Altogether, the Atoh1 CRE knock-in mouse strain might be a useful tool to explore the function and connectivity of MNs involved in fine motor control when combined with other genetic or viral strategies that can restrict labeling specifically to the IH and IF MNs. Accordingly, we provide an example of sparse labeling of IH and IF MNs using an intersectional genetic approach.