Astrocyte ensheathment of calyx-forming axons of the auditory brainstem precedes accelerated expression of myelin genes and myelination by oligodendrocytes.

Early postnatal brain development involves complex interactions among maturing neurons and glial cells that drive tissue organization. We previously analyzed gene expression in tissue from the mouse medial nucleus of the trapezoid body (MNTB) during the first postnatal week to study changes that surround rapid growth of the large calyx of Held (CH) nerve terminal. Here, we present genes that show significant changes in gene expression level during the second postnatal week, a developmental timeframe that brackets the onset of airborne sound stimulation and the early stages of myelination. Gene Ontology analysis revealed that many of these genes are related to the myelination process. Further investigation of these genes using a previously published cell type-specific bulk RNA-Seq data set in cortex and our own single-cell RNA-Seq data set in the MNTB revealed enrichment of these genes in the oligodendrocyte lineage (OL) cells. Combining the postnatal day (P)6-P14 microarray gene expression data with the previously published P0-P6 data provided fine temporal resolution to investigate the initiation and subsequent waves of gene expression related to OL cell maturation and the process of myelination. Many genes showed increasing expression levels between P2 and P6 in patterns that reflect OL cell maturation. Correspondingly, the first myelin proteins were detected by P4. Using a complementary, developmental series of electron microscopy 3D image volumes, we analyzed the temporal progression of axon wrapping and myelination in the MNTB. By employing a combination of established ultrastructural criteria to classify reconstructed early postnatal glial cells in the 3D volumes, we demonstrated for the first time that astrocytes within the mouse MNTB extensively wrap the axons of the growing CH terminal prior to OL cell wrapping and compaction of myelin. Our data revealed significant expression of several myelin genes and enrichment of multiple genes associated with lipid metabolism in astrocytes, which may subserve axon wrapping in addition to myelin formation. The transition from axon wrapping by astrocytes to OL cells occurs rapidly between P4 and P9 and identifies a potential new role of astrocytes in priming calyceal axons for subsequent myelination.

Dysregulation of astrocyte-secreted pleiotrophin contributes to neuronal structural and functional deficits in Down Syndrome.

Neuronal dendrite patterning and synapse formation are tightly regulated during development to promote proper connectivity. Astrocyte-secreted proteins act as guidance and pro-synaptogenic factors during development, but little is known about how astrocytes may contribute to neurodevelopmental disorders. Here we identify down-regulation of the astrocyte-secreted molecule pleiotrophin as a major contributor to neuronal morphological alterations in the Ts65Dn mouse model of Down Syndrome. We find overlapping deficits in neuronal dendrites, spines and intracortical synapses in Ts65Dn mutant and pleiotrophin knockout mice. By targeting pleiotrophin overexpression to astrocytes in adult Ts65Dn mutant mice in vivo , we show that pleiotrophin can rescue dendrite morphology and spine density and increase excitatory synapse number. We further demonstrate functional improvements in behavior. Our findings identify pleiotrophin as a molecule that can be used in Down Syndrome to promote proper circuit connectivity, importantly at later stages of development after typical periods of circuit refinement have completed.

Astrocyte contribution to dysfunction, risk and progression in neurodegenerative disorders.

There is increasing appreciation that non-neuronal cells contribute to the initiation, progression and pathology of diverse neurodegenerative disorders. This Review focuses on the role of astrocytes in disorders including Alzheimer disease, Parkinson disease, Huntington disease and amyotrophic lateral sclerosis. The important roles astrocytes have in supporting neuronal function in the healthy brain are considered, along with studies that have demonstrated how the physiological properties of astrocytes are altered in neurodegenerative disorders and may explain their contribution to neurodegeneration. Further, the question of whether in neurodegenerative disorders with specific genetic mutations these mutations directly impact on astrocyte function, and may suggest a driving role for astrocytes in disease initiation, is discussed. A summary of how astrocyte transcriptomic and proteomic signatures are altered during the progression of neurodegenerative disorders and may relate to functional changes is provided. Given the central role of astrocytes in neurodegenerative disorders, potential strategies to target these cells for future therapeutic avenues are discussed.

Transcriptional profiling reveals roles of intercellular Fgf9 signaling in astrocyte maturation and synaptic refinement during brainstem development.

Neural tissue maturation is a coordinated process under tight transcriptional control. We previously analyzed the kinetics of gene expression in the medial nucleus of the trapezoid body (MNTB) in the brainstem during the critical postnatal phase of its development. While this work revealed timed execution of transcriptional programs, it was blind to the specific cells where gene expression changes occurred. Here, we utilized single-cell RNA-Seq to determine transcriptional profiles of each major MNTB cell type. We discerned directional signaling patterns between neuronal, glial, and vascular-associated cells for VEGF, TGFß, and Delta-Notch pathways during a robust period of vascular remodeling in the MNTB. Furthermore, we describe functional outcomes of the disruption of neuron-astrocyte fibroblast growth factor 9 (Fgf9) signaling. We used a conditional KO (cKO) approach to genetically delete Fgf9 from principal neurons in the MNTB, which led to an early onset of glial fibrillary acidic protein (Gfap) expression in astrocytes. In turn, Fgf9 cKO mice show increased levels of astrocyte-enriched brevican (Bcan), a component of the perineuronal net matrix that ensheaths principal neurons in the MNTB and the large calyx of Held terminal, while levels of the neuron-enriched hyaluronan and proteoglycan link protein 1 (Hapln1) were unchanged. Finally, volumetric analysis of vesicular glutamate transporters 1 and 2 (Vglut1/2), which serves as a proxy for terminal size, revealed an increase in calyx of Held volume in the Fgf9 cKO. Overall, we demonstrate a coordinated neuron-astrocyte Fgf9 signaling network that functions to regulate astrocyte maturation, perineuronal net structure, and synaptic refinement.

miRNA-378a as a key regulator of cardiovascular health following engineered nanomaterial inhalation exposure.

Nano-titanium dioxide (nano-TiO2), though one of the most utilized and produced engineered nanomaterials (ENMs), diminishes cardiovascular function through dysregulation of metabolism and mitochondrial bioenergetics following inhalation exposure. The molecular mechanisms governing this cardiac dysfunction remain largely unknown. The purpose of this study was to elucidate molecular mediators that connect nano-TiO2 exposure with impaired cardiac function. Specifically, we were interested in the role of microRNA (miRNA) expression in the resulting dysfunction. Not only are miRNA global regulators of gene expression, but also miRNA-based therapeutics provide a realistic treatment modality. Wild type and MiRNA-378a knockout mice were exposed to nano-TiO2 with an aerodynamic diameter of 182 ± 1.70 nm and a mass concentration of 11.09 mg/m3 for 4 h. Cardiac function, utilizing the Vevo 2100 Imaging System, electron transport chain complex activities, and mitochondrial respiration assessed cardiac and mitochondrial function. Immunoblotting and qPCR examined molecular targets of miRNA-378a. MiRNA-378a-3p expression was increased 48 h post inhalation exposure to nano-TiO2. Knockout of miRNA-378a preserved cardiac function following exposure as revealed by preserved E/A ratio and E/SR ratio. In knockout animals, complex I, III, and IV activities (∼2- to 6-fold) and fatty acid respiration (∼5-fold) were significantly increased. MiRNA-378a regulated proteins involved in mitochondrial fusion, transcription, and fatty acid metabolism. MiRNA-378a-3p acts as a negative regulator of mitochondrial metabolic and biogenesis pathways. MiRNA-378a knockout animals provide a protective effect against nano-TiO2 inhalation exposure by altering mitochondrial structure and function. This is the first study to manipulate a miRNA to attenuate the effects of ENM exposure.

Glial Cell Expansion Coincides with Neural Circuit Formation in the Developing Auditory Brainstem.

Neural circuit formation involves maturation of neuronal, glial and vascular cells, as well as cell proliferation and cell death. A fundamental understanding of cellular mechanisms is enhanced by quantification of cell types during key events in synapse formation and pruning and possessing qualified genetic tools for cell type-specific manipulation. Acquiring this information in turn requires validated cell markers and genetic tools. We quantified changing proportions of neurons, astrocytes, oligodendrocytes, and microglia in the medial nucleus of the trapezoid body (MNTB) during neural circuit development. Cell type-specific markers, light microscopy and 3D virtual reality software, the latter developed in our laboratory, were used to count cells within distinct cell populations at postnatal days (P)3 and P6, bracketing the period of nerve terminal growth and pruning in this system. These data revealed a change from roughly equal numbers of neurons and glia at P3 to a 1.5:1 ratio of glia to neurons at P6. PCNA and PH3 labeling revealed that proliferation of oligodendrocytes contributed to the increase in glial cell number during this timeframe. We next evaluated Cre driver lines for selectivity in labeling cell populations. En1-Cre was specific for MNTB neurons. PDGFRα-Cre and Aldh1L1-Cre, thought to be mostly specific for oligodendrocyte lineage cells and astrocytes, respectively, both labeled significant numbers of neurons, oligodendrocytes, and astrocytes and are non-specific genetic tools in this neural system.

Temporal patterns of gene expression during calyx of held development.

Relating changes in gene expression to discrete developmental events remains an elusive challenge in neuroscience, in part because most neural territories are comprised of multiple cell types that mature over extended periods of time. The medial nucleus of the trapezoid body (MNTB) is an attractive vertebrate model system that contains a nearly homogeneous population of neurons, which are innervated by large glutamatergic nerve terminals called calyces of Held (CH). Key steps in maturation of CHs and MNTB neurons, including CH growth and competition, occur very quickly for most cells between postnatal days (P)2 and P6. Therefore, we characterized genome-wide changes in this system, with dense temporal sampling during the first postnatal week. We identified 541 genes whose expression changed significantly between P0-6 and clustered them into eight groups based on temporal expression profiles. Candidate genes from each of the eight profile groups were validated in separate samples by qPCR. Our tissue sample permitted comparison of known glial and neuronal transcripts and revealed that monotonically increasing or decreasing expression profiles tended to be associated with glia and neurons, respectively. Gene ontology revealed enrichment of genes involved in axon pathfinding, cell differentiation, cell adhesion and extracellular matrix. The latter category included elements of perineuronal nets, a prominent feature of MNTB neurons that is morphologically distinct by P6, when CH growth and competition are resolved onto nearly all MNTB neurons. These results provide a genetic framework for investigation of general mechanisms responsible for nerve terminal growth and maturation.