Identification of a Unique TGF-β Dependent Molecular and Functional Signature in Microglia

Microglia are myeloid cells of the central nervous system (CNS) that participate both in normal CNS function and disease. We investigated the molecular signature of microglia and identified 239 genes and 8 microRNAs that were uniquely or highly expressed in microglia vs. myeloid and other immune cells. Out of 239 genes, 106 were enriched in microglia as compared to astrocytes, oligodendrocytes and neurons. This microglia signature was not observed in microglial lines or in monocytes recruited to the CNS and was also observed in human microglia. Based on this signature, we found a crucial role for TGF-β in microglial biology that included: 1) the requirement of TGF-β for the in vitro development of microglia that express the microglial molecular signature characteristic of adult microglia; and 2) the absence of microglia in CNS TGF-β1 deficient mice. Our results identify a unique microglial signature that is dependent on TGF-β signaling which provides insights into microglial biology and the possibility of targeting microglia for the treatment of CNS disease

Identification of Molecular Determinants of Testosterone-associated Angiogenesis and Neuronal Survival in Adult Songbird Forebrain

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Neuroscience Graduate Program, 2012.The HVC of the adult songbird forebrain undergoes gonadal steroid-mediated survival of new neurons throughout life. Angiogenesis and neuronal recruitment are serially and causally linked in this process, such that testosterone-induced angiogenesis is necessary for neuronal recruitment. To better understand the contribution of testosterone-induced transcriptional programs to this adult neuronal recruitment, we assessed the effects of androgen exposure on gene expression in the adult canary HVC. We first generated and sequenced a library of cDNAs derived from testosterone-treated adult female and male canary HVCs. We then used these sequences to establish two microarray platforms--a spotted cDNA array and a high-density oligonucleotide microarray. Using the higher quality high-density oligonucleotide array, we profiled the mRNAs of laser capture microdissected HVC sections, derived from testosterone-treated female canaries killed at varying time-points after androgen treatment. This analysis revealed >1000 probe sets that were regulated by testosterone; gene ontology analysis indicated strong differential regulation of genes associated with neuronal survival, while pathway analysis revealed strong differential expression of genes involved in nitric oxide signaling. In situ hybridization (ISH) confirmed that NRGN, RGS4, PPP3CA and CAMK2B mRNA were highly differentially expressed in HVC, whereas PLCB1 mRNA was diminished. Real-time quantitative PCR analysis confirmed that testosterone induced HVC RGS4 mRNA expression, but suppressed HVC NRGN, calcineurin/PPP3CA and CAMK2B. This pattern of regulation suggests that the nitric oxide regulatory pathways are critically involved in the androgenic modulation of cellular plasticity in the adult HVC, since this set of transcripts is highly associated with NO synthesis and signaling. In addition, we noted that the angiogenic modulators CRHBP and ADD3 were selectively enriched in the HVC; ISH then revealed that CRHBP mRNA expression was stimulated by testosterone specifically in HVC interneurons, while RGS4 was expressed in a complementary fashion in HVC excitatory projection neurons. Together, our transcriptional data suggest that HVC interneurons may subserve a regulatory role in testosterone-dependent angiogenesis, which may in turn govern HVC neuronal recruitment through NO-modulatory pathways

Double fluorescence in situ hybridization in fresh brain sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure. © JoVE 2006-2011 All Rights Reserved

Molecularly defined cortical astroglia subpopulation modulates neurons via secretion of Norrin

Despite expanding knowledge regarding the role of astroglia in regulating neuronal function, little is known about regional or functional subgroups of brain astroglia and how they may interact with neurons. We use an astroglia-specific promoter fragment in transgenic mice to identify an anatomically defined subset of adult gray matter astroglia. Using transcriptomic and histological analyses, we generate a combinatorial profile for the in vivo identification and characterization of this astroglia subpopulation. These astroglia are enriched in mouse cortical layer V; express distinct molecular markers, including Norrin and leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6), with corresponding layer-specific neuronal ligands; are found in the human cortex; and modulate neuronal activity. Astrocytic Norrin appears to regulate dendrites and spines; its loss, as occurring in Norrie disease, contributes to cortical dendritic spine loss. These studies provide evidence that human and rodent astroglia subtypes are regionally and functionally distinct, can regulate local neuronal dendrite and synaptic spine development, and contribute to disease