J. Ronald Doucette

Transcriptional control of oligodendrogenesis.

Oligodendrocytes (OGs) assemble the myelin sheath around axons in the central nervous system. Specification of cells into the OG lineage is largely the result of interplay between bone morphogenetic protein, sonic hedgehog and Notch signaling pathways, which regulate expression of transcription factors (TFs) dictating spatial and temporal aspects of oligodendrogenesis. Many of these TFs and others then direct OG development through to a mature myelinating OG. Here we describe signaling pathways and TFs that are inductive, inhibitory, and/or permissive to OG specification and maturation. We develop a basic transcriptional network and identify similarities and differences between regulation of oligodendrogenesis in the spinal cord and brain.

Sirt2 is a novel in vivo downstream target of Nkx2.2 and enhances oligodendroglial cell differentiation

Although Sirt2 is primarily expressed in oligodendrocytes of the central nervous system, its role in oligodendroglial lineage differentiation is not fully understood. Our findings demonstrate that the transcription factor Nkx2.2 binds to the Sirt2 promoter via histone deacetylase 1 (HDAC-1), the binding site for Nkx2.2 maps close to the start codon of the Sirt2 gene, and Nkx2.2 negatively regulates Sirt2 expression in CG4 cells, an oligodendroglial precursor cell line. HDAC-1 knock-down not only significantly attenuates the binding capacity of Nkx2.2 to the Sirt2 promoter but also releases repression of Sirt2 expression by Nkx2.2. Nkx2.2 over-expression down-regulates Sirt2 expression and delays differentiation of CG4 cells; in contrast, up-regulation of Sirt2 does not impact Nkx2.2 expression level. Sirt2 knock-down via RNAi or inhibition of Sirt2 by sirtinol, a Sirt2 activity inhibitor, blocks CG4 cell differentiation. Over-expression of Sirt2 facilitates CG4 cell differentiation at both molecular and cellular levels, enhancing expression of myelin basic protein and facilitating the growth of cell processes. We have conclusively demonstrated that Sirt2 enhances CG4 oligodendroglial differentiation and report a novel mechanism through which Nkx2.2 represses CG4 oligodendroglial differentiation via Sirt2.

Transcriptional Regulation of Neurogenesis in the Olfactory Epithelium

1. The olfactory epithelium (OE) is a simple structure that gives rise to olfactory sensory neurons (OSNs) throughout life.2. Numerous transcription factors (TFs) are expressed in regions of the OE which contain progenitor cells and OSNs. The function of some of these TFs in OSN development has been elucidated with the aide of transgenic knockout mice.3. We review here the current state of knowledge on the role of TFs in OE neurogenesis and relate the expression of these TFs, where possible, to the well-documented phenotype of the cells as they progress through the OSN lineage from progenitor cells to mature neurons.

Coordinated expression of Hoxa2, Hoxd1 and Pax6 in the developing diencephalon.

Coordinated expression of Hoxa2, Hoxd1 and Pax6 proteins were found to coincide with the three developmental stages of the diencephalon, as described for the mouse brain [4]. In the first stage (embryonic day (E) 10–12) Hoxa2, Hoxd1 and Pax6 (an early marker gene of the diencephalon) were expressed as early as E10.5 in prosomeres (p), p2 and p3. All three proteins continue to exhibit overlapping domains of expression at E12.5–13 (beginning of the second stage) when the primitive dense cell layer begins to differentiate into the internal germinal, external germinal and mantle layers. Towards the end of the second stage (E15), Pax6 expression was down- regulated whereas Hoxa2 and Hoxd1 continued to exhibit overlapping domains of expression for both protein and mRNA. Hoxd1 expression decreased significantly in the third stage of diencephalic development (E16–postnatal) such that only Hoxa2 expression persisted in the diencephalon of newborn mice. The temporal and spatial expression of these three proteins imply that coordinated waves of Hoxa2, Hoxd1 and Pax6 expression may be required to provide positional information for the specification of the diencephalon.

Hoxb4 in Oligodendrogenesis

Abstract1. Although recent advances have provided insight into the transcriptional control of oligodendrocyte (OG) development, little information exists on the role of clustered Hox genes in this process. The aim of this study was to examine the expression profile of Hoxb4 in the oligodendroglial lineage.2. Immunocytochemical analysis of primary mixed glial cultures demonstrated that Hoxb4 was expressed throughout OG development, being coexpressed with oligodendroglial markers, A2B5, O4 (97%), GalC (91%), and MBP (93%).3. Immunohistochemical analysis of transverse spinal cord sections demonstrated diffuse expression of Hoxb4 throughout the spinal cord at E12.5 (C16/T19), after which expression was confined primarily to the presumptive gray matter.4. At E14.25 (C19+/T21), Olig2+ cells had begun to migrate out from the ventral ventricular zone into the presumptive gray matter. These results suggest that Olig2+ cells could coexpress Hoxb4 since it is expressed throughout this region.5. The expression of Hoxb4 by cells of the OG lineage indicates that it could play a role in OG maturation.

Early Stages of Oligodendrocyte Development in the Embryonic Murine Spinal Cord Proceed Normally in the Absence of Hoxa2

Recent discoveries have enhanced our knowledge of the transcriptional control of oligodendrocyte (OG) development. In particular, the transcription factors (TFs) Olig2, Pax6, and Nkx2.2 have been shown to be important in the specification and/or maturation of the OG lineage. Although numerous other TFs are expressed by OGs, little is known regarding their role(s) in oligodendrogenesis. One such TF is the homeobox gene Hoxa2, which was recently shown to be expressed by O4+ pro‐oligodendrocytes. The objectives of this study were to examine the expression of Hoxa2 during the early stages of OG development, as well as to determine whether Hoxa2 is required for specification and/or early maturation of OGs. Immunocytochemical analysis of primary mixed glial cultures demonstrated that Hoxa2 was expressed throughout oligodendrogenesis, diminishing only with the acquisition of a myelinating phenotype. Serial transverse spinal cord sections from embryonic days 12.5, 14.25, 16, and 18 Hoxa2+/+, Hoxa2+/−, and Hoxa2−/− mice were subjected to single and double immunohistochemical analysis in order to examine Hoxa2, Olig2, Nkx2.2, and Pax6 expression profiles. Results obtained from Hoxa2+/+ and Hoxa2+/− mice suggested that Hoxa2 was expressed by migratory oligodendroglial cells. In addition, comparison of spinal cord sections obtained from Hoxa2+/+, Hoxa2+/−, and Hoxa2−/− mice suggested that specification and early maturation of OGs proceeded normally in the absence of Hoxa2, since there were no obvious alterations in the expression patterns of Olig2, Nkx2.2, and/or Pax6. Hence, although Hoxa2 is expressed throughout OG development, it does not appear to be critical for early stages of oligodendrogenesis in the murine spinal cord.

A novel method of eliminating non-neuronal proliferating cells from cultures of mouse dorsal root ganglia.

Abstract1. We hypothesized that non-neuronal cells could be eliminated from primary dorsal root ganglion (DRG) cultures by including a DNA topoisomerase inhibitor (camptothecin) during culture.2. Exposure to 20 μM camptothecin for 48 h, beginning at 3 days in vitro, reliably eliminates proliferating non-neuronal cells.3. Following camptothecin treatment, neurons survived and continued to extend neurites for several weeks without obvious defects in morphology or viability.4. Transient camptothecin exposure is therefore an efficient and fast-acting method to purify DRG neurons in culture.

RNA-binding Protein Quaking StabilizesSirt2mRNA during Oligodendroglial Differentiation

Myelination is controlled by timely expression of genes involved in the differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes (OLs). Sirtuin 2 (SIRT2), a NAD+-dependent deacetylase, plays a critical role in OL differentiation by promoting both arborization and downstream expression of myelin-specific genes. However, the mechanisms involved in regulating SIRT2 expression during OL development are largely unknown. The RNA-binding protein quaking (QKI) plays an important role in myelination by post-transcriptionally regulating the expression of several myelin specific genes. In quaking viable (qkv/qkv) mutant mice, SIRT2 protein is severely reduced; however, it is not known whether these genes interact to regulate OL differentiation. Here, we report for the first time that QKI directly binds to Sirt2 mRNA via a common quaking response element (QRE) located in the 3′ untranslated region (UTR) to control SIRT2 expression in OL lineage cells. This interaction is associated with increased stability and longer half-lives of Sirt2.1 and Sirt2.2 transcripts leading to increased accumulation of Sirt2 transcripts. Consistent with this, overexpression of qkI promoted the expression of Sirt2 mRNA and protein. However, overexpression of the nuclear isoform qkI-5 promoted the expression of Sirt2 mRNA, but not SIRT2 protein, and delayed OL differentiation. These results suggest that the balance in the subcellular distribution and temporal expression of QKI isoforms control the availability of Sirt2 mRNA for translation. Collectively, our study demonstrates that QKI directly plays a crucial role in the post-transcriptional regulation and expression of Sirt2 to facilitate OL differentiation.

Conditional Tet-Regulated Over-Expression of Hoxa2 in CG4 Cells Increases Their Proliferation and Delays Their Differentiation into Oligodendrocyte-like Cells Expressing Myelin Basic Protein

Hoxa2 gene was reported to be expressed by oligodendrocytes (OLs) and down-regulated at the terminal differentiation stage during oligodendrogenesis in mice (Nicolay et al. 2004b). To further investigate the role of Hoxa2 in oligodendroglial development, a tetracycline regulated controllable expression system was utilized to establish a stable cell line (CG4-SHoxa2 [sense Hoxa2]), where the expression level of Hoxa2 gene could be up-regulated. The impact of Hoxa2 over-expression on the proliferation and differentiation of CG4-SHoxa2 cells was investigated. Up-regulation of Hoxa2 increased the proliferation of CG4-SHoxa2 cells. The mRNA levels of PDGFαR (platelet-derived growth factor [PDGF] alpha receptor), which is expressed by OL progenitor cells, were not different in CG4-SHoxa2 cells compared to wild-type CG4 cells. Semi-quantitative RT-PCR revealed that the mRNA levels of myelin basic protein (MBP) was lower in CG4-SHoxa2 cells than in wild-type CG4 cells indicating the differentiation of CG4-SHoxa2 cells was delayed when the Hoxa2 gene was up-regulated.

Pharmacogenomics of interferon-β in multiple sclerosis: what has been accomplished and how can we ensure future progress?

Multiple sclerosis (MS) is a progressive disorder of the central nervous system, often resulting in significant disability in early adulthood. The field of pharmacogenomics holds promise in distinguishing responders from non-responders to drug treatment. Most studies on genetic polymorphisms in MS have addressed treatment with interferon-β, yet few findings have been replicated. This review outlines the barriers that currently hinder the validity, reproducibility, and inter-study comparison of pharmacogenomics research as it relates to the use of interferon-β. Notably, statistical power, varying definitions of responder status, varying assay and genotyping methodologies, and anti-interferon-β neutralizing antibodies significantly confound existing data. Future work should focus on addressing these factors in order to optimize interferon-β treatment outcomes in MS.