Does Sirt2 Regulate Cholesterol Biosynthesis During Oligodendroglial Differentiation In Vitro and In Vivo?

Sirtuin2 (SIRT2) is a deacetylase enzyme predominantly expressed in myelinating glia of the central nervous system (CNS). We have previously demonstrated that Sirt2 expression enhances oligodendrocyte (OL) differentiation and arborization in vitro, but the molecular targets of SIRT2 in OLs remain speculative. SIRT2 has been implicated in cholesterol biosynthesis by promoting the nuclear translocation of sterol regulatory element binding protein (SREBP)-2. We investigated this further in CNS myelination by examining the role of Sirt2 in cholesterol biosynthesis in vivo and in vitro employing Sirt2 -/- mice, primary OL cells and CG4-OL cells. Our results demonstrate that expression of cholesterol biosynthetic genes in the CNS white matter or cholesterol content in purified myelin fractions did not differ between Sirt2 -/- and age-matched wild-type mice. Cholesterol biosynthetic gene expression profiles and total cholesterol content were not altered in primary OLs from Sirt2 -/- mice and in CG4-OLs when Sirt2 was either down-regulated with RNAi or overexpressed. In addition, Sirt2 knockdown or overexpression in CG4-OLs had no effect on SREBP-2 nuclear translocation. Our results indicate that Sirt2 does not impact the expression of genes encoding enzymes involved in cholesterol biosynthesis, total cholesterol content, or nuclear translocation of SREBP-2 during OL differentiation and myelination.

Organotypic Cultures from the Adult CNS: A Novel Model to Study Demyelination and Remyelination Ex Vivo.

Experimental models of multiple sclerosis (MS) have significantly advanced our understanding of pathophysiology and therapeutic interventions. Although in vivo rodent models are considered to most closely represent the complex cellular and molecular disease states of the human central nervous system (CNS), these can be costly to maintain and require long timelines. Organotypic slice cultures maintain the cytotypic organization observed in the intact CNS, yet provide many of the experimental advantages of in vitro cell culture models. Cerebellar organotypic cultures have proven useful for studying myelination and remyelination, but this model has only been established using early postnatal tissue. This young brain tissue allows for neuro development ex vivo to mimic the 'mature' CNS; however, there are many differences between postnatal and adult organotypic cultures. This may be particularly relevant to MS, as a major barrier to myelin regeneration is age. This paper describes a modified protocol to study demyelination and remyelination in adult cerebellar tissue, which has been used to demonstrate neuroprotection with omega-3 fatty acids. Thus, adult cerebellar organotypic cultures provide a novel ex vivo platform for screening potential therapies in myelin degeneration and repair.

RNA-binding Protein Quaking Stabilizes Sirt2 mRNA 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.

Advances in the treatment of relapsing-remitting multiple sclerosis: the role of pegylated interferon ß-1a.

Multiple sclerosis (MS) is a progressive, neurodegenerative disease with unpredictable phases of relapse and remission. The cause of MS is unknown, but the pathology is characterized by infiltration of auto-reactive immune cells into the central nervous system (CNS) resulting in widespread neuroinflammation and neurodegeneration. Immunomodulatory-based therapies emerged in the 1990s and have been a cornerstone of disease management ever since. Interferon ß (IFNß) was the first biologic approved after demonstrating decreased relapse rates, disease activity and progression of disability in clinical trials. However, frequent dosing schedules have limited patient acceptance for long-term therapy. Pegylation, the process by which molecules of polyethylene glycol are covalently linked to a compound, has been utilized to increase the half-life of IFNß and decrease the frequency of administration required. To date, there has been one clinical trial evaluating the efficacy of pegylated IFN. The purpose of this article is to provide an overview of the role of IFN in the treatment of MS and evaluate the available evidence for pegylated IFN therapy in MS.

Protocols for Cloning, Expression, and Functional Analysis of Sirtuin2 (SIRT2).

SIRT2 is a NAD(+)-dependent deacetylase that belongs to the sirtuin family, which is comprised of seven members (SIRT1-SIRT7) in humans. Furthermore, recent study shows that the Sirt2 gene has three transcript variants in mice. Several diverse proteins have been identified as SIRT2 substrates. SIRT2 activity involves multiple cell processes including growth, differentiation, and energy metabolism. However, little is known of SIRT2's role in oligodendrocytes or in the myelin sheath, where it is an important component. Here we describe procedures that detail Sirt2 gene cloning, identification, expression, and biological analysis in cultured cells.

Pharmacogenomics of interferon-ß 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-ß, 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-ß. Notably, statistical power, varying definitions of responder status, varying assay and genotyping methodologies, and anti-interferon-ß neutralizing antibodies significantly confound existing data. Future work should focus on addressing these factors in order to optimize interferon-ß treatment outcomes in MS.

Current developments in pharmacogenomics of multiple sclerosis.

Pharmacogenomics has a significant potential to impact how we treat diseases. It involves targeting genetically identifiable populations with therapeutic interventions that promises to yield immediate positive health outcomes with lower or no side effects. The 'trial and error' method of treatment will no longer be necessary with the successful implementation of personalized medicine. The following is an overview of some new developments in pharmacogenomics of multiple sclerosis, and how it has the potential to improve future treatment.

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.

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.

Age-related and cuprizone-induced changes in myelin and transcription factor gene expression and in oligodendrocyte cell densities in the rostral corpus callosum of mice.

During aging, there is a decrease both in the stability of central nervous system (CNS) myelin once formed and in the efficiency of its repair by oligodendrocytes (OLs). To study CNS remyelination during aging, we used the cuprizone (a copper chelator) mouse model. Inclusion of cuprizone in the diet kills mature OLs and demyelinates axons in the rostral corpus callosum (CC) of mice, which enabled us to characterize age-related changes (i.e., 2-16 months of age) in glial cell response during the recruitment (i.e., demyelination) and differentiation (i.e., remyelination) phases of myelin repair. We have found that the time between 12 and 16 months of age is a critical period during which there is an age-related decrease in the number of OL lineage cells (Olig2(Nuc)+ve/GFAP-ve cells) in the rostral CC of both control mice and mice recovering from cuprizone-induced demyelination. Our results also show there was an age-related impaired recruitment of progenitor cells to replace lost OLs in spite of there being no major age-related decrease in the size of the progenitor cell pool (PDGFalphaR+ve/GFAP-ve, and Olig2(Nuc) +ve/PDGFalphaR+ve cells). However, there were cuprizone-induced increased numbers of astrocyte progenitor cells (Olig2(Cyto)+ve/PDGFalphaR+ve) in these same mice; thus PDGFalphaR+ve progenitor cells in mice as old as 16 months of age retain the ability to differentiate into astrocytes, with this fate choice occurring following cytoplasmic translocation of Olig2. These data reveal for the first time age-related differences in the differentiation of PDGFalphaR+ve progenitor cells into OLs and astrocytes and lead us to suggest that during aging there must be a transcriptional switch mechanism in the progenitor cell fate choice in favor of astrocytes. This may at least partially explain the age-related decrease in efficiency of OL myelination and remyelination.

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.

Hoxd1 is expressed by oligodendroglial cells and binds to a region of the human myelin oligodendrocyte glycoprotein promoter in vitro.

(1) Little information exists on the role of clustered Hox genes in oligodendrocyte (OG) development. This study examines the expression profile of Hoxd1 and identifies a potential downstream target in the OG lineage. (2) Immunocytochemical analysis of primary mixed glial cultures demonstrated Hoxd1 was expressed throughout OG development. (3) A human myelin protein gene, myelin oligodendrocyte glycoprotein (MOG), was identified as a putative downstream target of Hoxdl through Genbank searches utilizing the Hoxdl homeodomain consensus binding sequence. (4) The dissociation coefficient constant (KD) and dissociation rate constant (kd) of the Hoxd1-MOG complex, determined using electrophoretic mobility shift assays (EMSAs), were estimated to be 1.9 x 10(-7) M and 1.3 x 10(-3) s(-1), respectively. The DNA-Hoxdl homeodomain complex had a half-life (t1/2) of 15 min. (5) Mutational analysis of Hoxd1-MOG complexes revealed the binding affinity of M1 (with mutation from (-1054)5'-TAAT-3'(-1051) to TACT within the consensus binding site) and M2 (with mutation from (-1054)5'-TAATTG-3'(-1049) to TAATCC within the consensus binding site) probes to the MOG promoter was severely affected. Thus the TAATTG core of the binding sequence appears important for Hoxd1 specificity. (6) Analysis of the involvement of TAAT sites adjacent to the consensus sequence in Hoxdl binding showed the binding affinity of the M3 probe was affected, but not as severely as the M1 and M2 probes. These in vitro results suggest the TTTAATTGTA sequence is involved in Hoxd1 binding to the MOG promoter but neighboring TAAT sites may also be needed. Thus, MOG may be a target of Hoxd1.

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.

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.

Hoxb4 in oligodendrogenesis.

1. 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.

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

1. 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 microM 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.