Intracortical astrocyte subpopulations defined by astrocyte reporter Mice in the adult brain.

Although historically regarded as a homogeneous cell population, astrocytes in different brain regions exhibit differences in their physiological properties, such as gap-junction coupling, glutamate uptake dynamics, and intracellular Ca2+ response. Recent in vivo RNA profiles have further demonstrated the molecular heterogeneity of astrocytes in the adult CNS. Astrocyte heterogeneity exists not only inter-regionally but also intra-regionally. Despite the characteristic laminal organization of cortical layers and multiple sources of radial glia progenitors for (astro)gliogenesis, the molecular profile and functional properties of astroglial subpopulations in the adult cerebral cortex remain essentially undefined. Using two astrocyte reporter mouse lines: eaat2-tdTomato and Bac aldh1l1-eGFP, we identified tdT- eGFP+ , tdTlow eGFP+ , and tdThigh eGFP+ astroglial subpopulations (in an approximate 1:7:2 ratio) within the cortex. The tdT- eGFP+ astrocyte population is selectively localized at layers I-II and exhibits increased resting membrane potential and membrane resistance but reduced functional expression of the potassium channel Kir4.1. We also isolated individual astrocyte subpopulations through fluorescence activated cell sorting (FACS) and examined their transcriptome differences by RNA-seq. We found that the whole-genome transcriptional profiles of tdT- eGFP+ astrocytes are drastically different from that of tdTlow eGFP+ and tdThigh eGFP+ astrocytes. Particularly, elevated levels of several nonastrocyte genes that are typically specific to other glial cells, such as mog, mobp, Iba1, and pdgfrα, are observed in tdT- eGFP+ astrocytes, suggesting a less-specific molecular identity of these astrocytes. Overall, our study has unveiled molecular differences between adult cortical astroglial subpopulations, shedding new light on understanding their unique functions in the adult cortex.

Molecular and Functional Properties of Regional Astrocytes in the Adult Brain.

The molecular signature and functional properties of astroglial subtypes in the adult CNS remain largely undefined. By using translational ribosome affinity purification followed by RNA-Seq, we profiled astroglial ribosome-associated (presumably translating) mRNAs in major cortical and subcortical brain regions (cortex, hippocampus, caudate-putamen, nucleus accumbens, thalamus, and hypothalamus) of BAC aldh1l1-translational ribosome affinity purification (TRAP) mice (both sexes). We found that the expression of astroglial translating mRNAs closely follows the dorsoventral axis, especially from cortex/hippocampus to thalamus/hypothalamus posteriorly. This region-specific expression pattern of genes, such as synaptogenic modulator sparc and transcriptional factors (emx2, lhx2, and hopx), was validated by qRT-PCR and immunostaining in brain sections. Interestingly, cortical or subcortical astrocytes selectively promote neurite growth and synaptic activity of neurons only from the same region in mismatched cocultures, exhibiting region-matched astrocyte to neuron communication. Overall, these results generated new molecular signature of astrocyte types in the adult CNS, providing insights into their origin and functional diversity.SIGNIFICANCE STATEMENT We investigated the in vivo molecular and functional heterogeneity of astrocytes inter-regionally from adult brain. Our results showed that the expression pattern of ribosome-associated mRNA profiles in astrocytes closely follows the dorsoventral axis, especially posteriorly from cortex/hippocampus to thalamus/hypothalamus. In line with this, our functional results further demonstrated region-selective roles of cortical and subcortical astrocytes in regulating cortical or subcortical neuronal synaptogenesis and maturation. These in vivo studies provide a previously uncharacterized and important molecular atlas for exploring region-specific astroglial functions.

Creation of a Psychotic Disorders Research Advisory Board as a Shared Resource.

Community engagement is important for research, yet many researchers do not routinely seek feedback from people with lived experience. A key barrier to this engagement is that the resources required to create an advisory board may be unavailable to individual investigators, and creating an advisory board for a single study may often be impractical. In this column, the authors describe how to create a standing research advisory board that can serve as a shared resource for researchers and community members and provide a psychosis research advisory board example to aid discussion.

Elevated exosomal secretion of miR-124-3p from spinal neurons positively associates with disease severity in ALS.

MicroRNAs (miRs) are powerful regulators of CNS development and diseases. Plasma and cerebrospinal fluid (CSF) miRs have recently been implicated as potential new sources for biomarker development. Previously we showed that miR-124-3p, an essential miR for neuronal identity, is highly abundant in neuronal exosomes and its expression decreases in spinal cord of ALS model SOD1G93A mice. In the current study, we found a disease associated reduction of miR-124-3p levels specifically in spinal neurons using in situ hybridization. By employing our recently developed exosome reporter mice in combination with sciatic nerve injections, we observed an increased association of miR-124-3p with spinal motor neuron-derived exosomes in SOD1G93A mice, even at the pre-symptomatic stage. Sciatic nerve injection delivered miR-124-3p is also more frequently localized outside of spinal motor neurons in SOD1G93A mice. Subsequent quantitative analysis of miR-124-3p levels in CSF exosomes from ALS patients found a significant correlation between CSF exosomal miR-124-3p levels and disease stage (indicated by the ALSFRS-R score) of (male) ALS patients. These results provide preliminary evidence to support the potential use of CSF exosomal miR-124-3p as a disease stage indicator in ALS.

Exosome reporter mice reveal the involvement of exosomes in mediating neuron to astroglia communication in the CNS.

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.

A transgenic resource for conditional competitive inhibition of conserved Drosophila microRNAs.

Although the impact of microRNAs (miRNAs) in development and disease is well established, understanding the function of individual miRNAs remains challenging. Development of competitive inhibitor molecules such as miRNA sponges has allowed the community to address individual miRNA function in vivo. However, the application of these loss-of-function strategies has been limited. Here we offer a comprehensive library of 141 conditional miRNA sponges targeting well-conserved miRNAs in Drosophila. Ubiquitous miRNA sponge delivery and consequent systemic miRNA inhibition uncovers a relatively small number of miRNA families underlying viability and gross morphogenesis, with false discovery rates in the 4-8% range. In contrast, tissue-specific silencing of muscle-enriched miRNAs reveals a surprisingly large number of novel miRNA contributions to the maintenance of adult indirect flight muscle structure and function. A strong correlation between miRNA abundance and physiological relevance is not observed, underscoring the importance of unbiased screens when assessing the contributions of miRNAs to complex biological processes.