10. Mouse Neurogenetics II: Chromatin Control of Development and Regeneration in Myelinating Glia

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1 10. Mouse Neurogenetics II: Chromatin Control of Development and Regeneration in Myelinating Glia 10.1 Introduction There are two types of myelinating cells: Oligodendrocytes in the central nervous system (CNS) that is composed of the brain and the spinal cord, and Schwann cells in the peripheral system (PNS), which consists in the autonomic nervous system that innervates the organs of the body and the somatic nervous systems that is under conscious control. Although oligodendrocytes and Schwann cells originate from different progenitors and differ in many ways, they share the common function of myelinating axons. Myelin is a lipid-rich structure that is found in vertebrates and in some invertebrates, and is necessary to insulate axons and to ensure saltatory nerve conduction. Without myelin, nerves are not functional Causes of demyelination and dysmyelination Loss of myelin - or demyelination - and aberrant myelin formation - or dysmyelination - can have different etiologies. Demyelination or dysmyelination can be due to autoimmune and/or inherited demyelinating diseases. In the CNS, the most common demyelinating disease is multiple sclerosis (MS) that has a strong inflammatory component and proceeds through attacks that specifically target oligodendrocytes. The first demyelinating lesions in MS patients are usually efficiently remyelinated, but as the disease progresses, remyelination becomes inefficient. This leads to permanent disability. Other less frequent demyelinating diseases include acute disseminated encephalomyelitis and transverse myelitis that are sometimes associated with MS, leukodystrophy, central pontine myelinolysis, glioma that are tumors due to proliferation of glial cells such as oligodendrocytes, and schizophrenia that has also been associated with demyelinating lesions. In the PNS, Charcot-Marie-Tooth diseases and the Waardenburg syndrome are inherited diseases that can lead to demyelination or dysmyelination. Demyelination can be also caused by inflammatory diseases such as the Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathies, and tumors called neurofibromatosis. Demyelination can also be secondary to diabetes, toxic agents, and always occurs more or less during aging. Finally, demyelination occurs after a trauma. While CNS lesions do not regenerate efficiently, PNS lesions can fully regenerate. In general, the prognosis of demyelinating and dysmyelinating diseases is rather poor, since there is a lack of efficient treatment and those lead to severe disabilities Schwann cell lineage As mentioned above, lesions of the PNS can fully regenerate. This is largely due to the peculiar ability of Schwann cells to de-differentiate and re-differentiate after a nerve lesion to foster axonal re-growth and remyelination. Oligodendrocytes do not 1

2 have this ability. Instead, remyelination in the CNS is achieved by oligodendrocytes precursor cells (OPCs) that migrate into the lesion site to remyelinate axons. However, the efficiency of this process is limited. Because of their exceptional regenerative properties, Schwann cells are very interesting cells to study, and understanding the molecular mechanisms of their cell cycle is key to their future use in regenerative medicine. Schwann cells originate from neural crest cells that also give rise to other cell types including sensory neurons, melanocytes, chondrocytes, and smooth-muscle cells. After the specification step into the glial lineage, Schwann cell precursors differentiate into immature Schwann cells that surround bundles of axons of different calibers. The next differentiation step determines whether a Schwann cell will become myelinating or non-myelinating. While small caliber axons remain in bundles associated with non-myelinating Schwann cells, large caliber axons are sorted into a one-to-one relationship with Schwann cells. This process, called radial sorting, leads to the pro-myelinating stage, where a Schwann cell forms one-and-a-half wraps around an axon, but no myelin yet. During the last differentiation step, Schwann cells produce a thick myelin sheath around sorted axons. The different steps of Schwann cell development are illustrated in Figure 1. Immature Promyelinating Myelinating Electron microscopy (morphology) Mutations in Krox20 gene lead to Charcot-Marie-Tooth disease Specification Neural crest cell Schwann cell precursor (Mouse) E11 E12-13 Radial Sorting Immature Schwann cell E15 Promyelinating Schwann cell Birth Myelination Myelinating Schwann cell Adult Figure 1. Schwann cell development Sox10, a member of the high mobility group of transcription factors, is expressed already in neural crest cells and is required for each step of the differentiation process, including the specification step. The process of Schwann cell differentiation is tightly regulated by intracellular and extracellular cues, and only when all necessary signals are perfectly orchestrated can peripheral nerves become fully functional. As mentioned before, Schwann cells originate from neural crest cells that are a population of migratory stem cells. These cells emigrate from the dorsal neural tube to form, among other structures, the dorsal root ganglia (DRG) and the peripheral nerves. Sensory neurons and Schwann cell precursors specify in the dorsal root ganglia. Schwann cells continue their journey by migrating into the peripheral nerves along axons, while the cell bodies of sensory neurons remain in the DRG, together with satellite cells that are also derived from the neural crest. Specification of sensory 2

3 neurons occurs in two waves, at embryonic day (E) 9.5 and E10 in mouse development, whereas specification of the Schwann cell lineage occurs later at E11. While Sox10 is downregulated in neurons, it remains highly expressed in the Schwann cell lineage. At E11, Sox10 is thus used as a marker of the glial lineage including Schwann cells in DRG and peripheral nerves, and can be detected either at the protein level by immunofluorescence or at the mrna level by in situ hybridization. Fabp7 (fatty-acid binding protein 7) and P0 (myelin protein zero) are other early markers of the Schwann cell lineage and can be well detected already at E11 at the mrna level. As for most key transcription factors, the Sox10 gene contains several enhancers in its non-coding region. These different enhancers are highly conserved among species and allow for spatiotemporal control of Sox10 expression. The distinct patterns of Sox10 enhancers activity have been shown in mouse embryos by in vivo beta-galactosidase reporter assay. Activity of the U3 enhancer, also called MCS4, is essential for Sox10 expression at the specification step into the glial lineage. Later on, another transcription factor, Krox20, is absolutely required for the myelination process that starts immediately after radial sorting at birth in mouse pups. Radial sorting is an interactive process between axons and Schwann cells. Axons express Neuregulin 1 (NRG1) at their surface. ErbB2 and ErbB3 are receptors for NRG1 and are expressed on the Schwann cell surface. High concentrations of NRG1 on the axonal surface instruct Schwann cells to sort axons. Big caliber axons express more NRG1 than small caliber axons, and will therefore be sorted in a one-to-one relationship with a Schwann cell (Figure 2). Non-myelinating Schwann cell Unsorted axons Sorted axons Figure 2. Axonal radial sorting Nave & Schwab, Nature Neuroscience (2005) Myelinating Schwann cells 3

4 Myelination is tightly linked to radial sorting. Indeed, only sorted axons will get myelinated and myelination starts right after radial sorting. Myelination is controlled by a network of transcription factors that act together to activate the transcription of Krox20 gene. Krox20 and Sox10 both activate the transcription of myelin genes (Figure 3). NFATc NFATc Figure 3. Transcriptional network of Schwann cell myelination Modified from Svaren and Meijer, Glia (2008) How are transcription factors controlled and activated? Extracellular cues such as NRG1 trigger the activation of intracellular pathways, such as AKT, which in turn lead to activation or upregulation of transcription factors. At the chromatin level, different architectures facilitate the access for transcription factors to DNA or in contrast restrict this access. We will focus the second part of this lecture on the involvement of chromatin-remodeling enzymes in the development of Schwann cells Chromatin remodeling Chromatin is a chain of nucleosomes. A nucleosome consists of 147 base pairs wrapped around a histone octamer: 2 x histone H2A, 2 x histone H2B, 2 x histone H3, and 2 x histone H4. Histone H1 is sometimes recruited to this structure to stabilize it further. This usually results in gene silencing and heterochromatin formation. Chromatin remodeling enzymes allow for dynamic changes of chromatin architecture. These changes can be achieved by DNA methylation or post-translational modifications of histones, such as acetylation, methylation, phosphorylation, ubiquitination, SUMOylation, and ADP-ribosylation. These modifications can either result in locally more condensed or more relaxed chromatin, rendering this region of DNA more or less accessible for the transcriptional machinery. We will focus on histone acetylation and deacetylation that are key histone modifications for the control of gene expression. Histone methylation is equally important, but we will not have the time to discuss this topic. Histone acetylation is achieved by histone acetyltransferases (HATs). As mentioned above, this process is dynamic and therefore reversible. Histone deacetylation is catalyzed by histone deacetylases (HDACs). HATs and HDACs target lysine 4

5 residues. These enzymes are classified into different families or classes, based on their structure. When histones are acetylated by HATs, their attraction to DNA is loosen. This results in a relaxed chromatin that facilitates DNA access for transcription factors and usually corresponds to a proliferation state. When histones are deacetylated by HDACs, their attraction to DNA is tight, and this leads to a condensed chromatin that limits or select DNA access for the transcriptional machinery, and usually corresponds to a differentiation state (Figure 4). Acetyl Ac groups Ac H4 H3 H2A H2B Ac Ac Histone deacetylases (HDACs) Histone acetyltransferases (HATs) Loosely wound H4 H3 H2A H2B Tightly wound Facilitated access for transcription factors Proliferation (usually) Figure 4. Outcome of histone acetylation and deacetylation Limited/selective access Differentiation (usually) HATs and HDACs acetylate and deacetylate histones, respectively, but they also have non-histone targets, such as transcription factors. Therefore, HATs and HDACs regulate transcriptional activity at different levels: by modifying chromatin architecture and by directly regulating the activity of transcription factors. Members of the HDAC family can have other enzymatic activities that also participate to chromatin remodeling, such as mono-adp ribosyltransferase and possibly SUMO E3 ligase. There are 18 known mammalian HDACs, subdivided into 4 classes. Class I (HDAC1, 2, 3, 8), class II (HDAC4, 5, 6, 7, 9, 10) and class IV (HDAC11) are the classical HDACs, whose activity is dependent on Zn 2+. Class III is a group of 7 enzymes (Sirt1-7) that are called sirtuins and are structurally unrelated to the classical HDACs. The activity of sirtuins depends on availability of NAD +. HDACs have multiple subcellular localizations, implying that they have multiple functions. Although all or most HDACs can theoretically deacetylate histones, their subcellular localization is not always compatible with this function. HDACs such as HDAC1 and HDAC2 are found in the nucleus, whereas other HDACs can be found in the cytoplasm or shuttle between the two compartments. Some sirtuins are even found in mitochondria and in the nucleolus (Figure 5). HDAC1 and HDAC2 have major functions in the regulation of gene expression. These two highly homologous HDACs are often found in the same multiprotein complexes. They do not have the ability to bind DNA directly, and they can therefore remodel chromatin only by interaction with DNA-binding proteins. 5

6 18 known mammalian HDACS 4 different classes (based on structure) Class I HDAC1 HDAC2 HDAC3 HDAC8 Class IV HDAC11 Class II HDAC4 HDAC5 HDAC6 HDAC7 HDAC9 HDAC10 Class III Sirt1-7 Sirtuins, NAD + -dependent Multiple subcellular localizations = multiple functions cytoplasm Classical HDACs Zn 2+ -dependent mitochondria HDAC8 HDAC1 and HDAC2 are highly homologous NLS Catalytic domain nucleus Figure 5. HDAC classifications and localizations nucleolus 10.5 Study of HDAC1 and HDAC2 functions in the development and maintenance of Schwann cells in mice To study the function of a gene in mice, the best way is usually to get rid of this gene and see what happens. To ablate a gene specifically in one tissue, we make use of the Cre/loxP system. We use mice carrying our gene of interest that has been modified by the insertion of two loxp sites flanking one or several exons that are absolutely necessary for the function of this gene. A gene with such modifications is "floxed". We cross mice carrying a floxed gene with mice carrying the Cre recombinase under control of a tissue-specific promoter (that is specifically active in the tissue of interest and not in other tissues). Offspring of these mice will express the Cre recombinase specifically in the tissue of interest. The Cre recombinase will recognize the two loxp sites and ablate whatever sequence located between these two loxp sites. This will result in loss of gene function only in the tissue of interest. This type of knockout (KO) mouse line is called a conditional KO (Figure 6). loxp loxp loxp Figure 6. Cell-specific gene ablation using the Cre/LoxP system 6

7 To ablate HDAC1 and HDAC2 specifically in neural crest cells, exon 6 containing the catalytic domain of HDAC1 and HDAC2 has been floxed and mice carrying these modified genes were generated. These mice were crossed with mice expressing the Cre recombinase under control of the Wnt1 promoter, active in neural crest cells starting from E8.5. Since HDAC1 and HDAC2 are highly homologous, it is usually necessary to ablate both genes for efficient loss of function. If only one of the two genes is ablated, the gene that is still expressed usually takes over the function of the ablated gene. In double HDAC1/HDAC2 mutants, neurogenesis is reduced, neuron migration is altered and there is an increased neuronal apoptosis. A massive apoptosis of smoothmuscle cells also occurs and there is a complete block of specification into the Schwann cell lineage. Double HDAC1/2 mutant embryos do not survive after E12.5, probably because of heart failure. It is possible to trace neural crest cells and their derivatives by crossing these mice with a reporter mouse line. Such a line carries a reporter gene, for example LacZ (codes for beta-galactosidase), under control of a ubiquitously active promoter and a floxed STOP cassette in between the promoter and the reporter gene. In the presence of the Cre rcombinase, the STOP cassette is excised and the reporter gene is expressed. To visualize neural crest cells and their derivatives, we detect beta-galactosidase activity by incubating whole embryos with X-Gal, a substrate of beta-galactosidase; this results in a blue staining of betagalactosidase-expressing cells (Figure 7). Neural Crest Cells active from E8.5 X E11.5 In vivo Fate mapping (Whole-mount XGal staining) Control Mutant (death at E12.5) Figure 7. In-vivo fate mapping using LacZ reporter mouse line To ablate HDAC1 and HDAC2 specifically in Schwann cells, there are different Cre lines available, either Dhh-Cre (Cre under control of the Dhh promoter) or P0-Cre (Cre under control of the P0 promoter). The Dhh promoter is active from E12.5 in Schwann cells. At this time-point, cells are already at the Schwann cell precursor stage. Double HDAC1/2 mutants are born, but rapidly develop a strong phenotype of tremor, reduced hind limb mobility, and die around postnatal day (P) 17. At P16, control sciatic nerves are well myelinated. They have a white color that is given by the lipids of the myelin sheath. In contrast, double mutant sciatic nerves are thin and translucent. This is a sign of severe hypomyelination. By electron microscopy, we can visualize the morphology of cells in the sciatic nerve. In double HDAC1/2 mutants, there is a radial sorting delay and indeed a severe hypomyelination. By TUNEL assay 7

8 or cleaved caspase 3 staining, we can detect apoptotic cells. Cleaved caspase 3 detects apoptotic cells at an early stage, whereas the TUNEL assay detects fragmented nuclei (late stage). To study the function of a gene in the maintenance of the peripheral nervous system or in its regeneration, we need to ablate this gene at the adult stage, when development is complete. For this purpose, we use a mouse line where Cre recombinase activity can be induced by tamoxifen, and we thus generate an inducible conditional KO mouse. To ablate HDAC1 and HDAC2 in Schwann cells in adult mice, there are two Cre mouse lines available: either the PLP-CreERT2 or P0Cx- CreERT2 mouse lines, where the Cre recombinase is expressed as a fusion protein with the hormone binding domain of the estrogen receptor (ER). In the absence of tamoxifen, HSP90 interacts with ER and thereby prevents the translocation of Cre into the nucleus. Upon tamoxifen injection, HSP90 is removed from the Cre-ER fusion protein that can translocate into the nucleus to excise the sequence between the two loxp sites in the gene of interest. In these mouse lines, the Cre recombinase is under control of the PLP promoter that is active in Schwann cells and also in oligodendrocytes, or under control of the P0 promoter that is active in Schwann cells only (Figure 8). Figure 8. Tamoxifen-inducible Cre recombinase system Six weeks after tamoxifen injection, double HDAC1/2 mutant mice develop hind limb weakness. By electron microscopy, we can detect many demyelinated axons, remyelinated axons (axons with a thin myelin sheath), and macrophages. The presence of remyelinated axons indicate that these axons have been demyelinated previously. Demyelination leads to accumulation of myelin debris in the nerve. This is a signal for macrophages to invade the nerve to clear out myelin debris. Myelin contains several inhibitors of axonal re-growth and it is therefore necessary that immune cells such as macrophages clear out the nerve from myelin debris to enable the regeneration process. To assess the loss of function in mice, it is possible to use behavioral analyses when the phenotype developed by mutant mice is strong enough. These behavioral tests include the RotaRod test (to assess coordination and endurance), the grip strength test (strength), gait analysis (coordination), the hot plate test (sensory function). These 8

9 tests can also be used to assess the recovery after a lesion. However, when the phenotype developed by the mice is not very strong, it is difficult to detect loss or gain of function with these behavioral tests. In that case, electrophysiology that measures the nerve conduction velocity will be the method of choice to assess loss or gain of function. The key steps of the regeneration process after a sciatic nerve crush lesion are well described in adult mice (Figure 9): - At 5 days post lesion, Schwann cells upregulate inhibitors of myelination that induce their de-differentiation and myelin breakdown. This leads to the invasion of the nerve by macrophages. In the meantime, injured axons degenerate by fragmentation up to the site of lesion, - At 12 days post lesion, axons have regrown, myelin debris have been alsmost all cleared by macrophages and Schwann cells, and Schwann cells upregulate inducers of myelination to start to re-differentiate, - At 1 month post lesion, Schwann cell express myelin proteins and all axons are remyelinated, but the myelin sheath has not reached a maximum thickness yet. Macrophages are at this point less abundant and actively leave the nerve - At 2 months post lesion, all myelin proteins are expressed by Schwann cells and all axons are re-myelinated to their maximum. A few macrophages can still be detected, but almost all of them are gone. The nerve is fully regenerated! Figure 9. Key steps of the regeneration process after a sciatic nerve lesion in adult mice At 5 days post lesion, Schwann cell de-differentiation and myelin breakdown are faster in double HDAC1/2 mutant mice compared to control mice. And at 1 month post lesion, remyelination is thinner in mutants compared to controls. These observations illustrate the involvement of HDACs in Schwann cells during the regeneration process Mechanistic analyses of HDAC1 and HDAC2 functions in Schwann cell development We have shown that HDAC1 and HDAC2 interact with the transcription factor Sox10 to bind to the Sox10 promoter and the Krox20 MSE (enhancer) and thereby activate the transcription of Sox10 and Krox20. The group of Chen et al. also found that HDAC1 and HDAC2 interact with NFkB to activate the Sox10 promoter. Sox10 increases the levels of active beta-catenin (ABC), which in turn increases also Sox10 and Krox20 levels. The function of this mechanism is most likely to enhance the myelination process. In addition, HDAC1 promotes Schwann cell survival by preventing precocious increase of ABC levels. 9

10 To identify a mechanism of action, it is often necessary to complement in vivo studies with cell culture methods. Advantages to use cell culture include the possibility to obtain a lot of material, working with a pure population of cells, reducing the number of experimental animals, and being able to easily manipulate expression levels of proteins of interest. Disadvantages are mainly due to the fact that the environment of the cells is different in culture compared to the in vivo environment, and thus cells in culture can behave differently than within a whole organism. Sometimes, it is difficult to mimic a physiologic process in vitro. Finally, working with a homogenous population of cells does not give the possibility to assess the variability of a process depending on genetic background. As a general rule, it is usually better to use primary cell cultures when possible because they usually behave similarly to the in vivo situation; which is not always the case with immortalized cell lines. For mechanistic analyses of neural crest specification, neural crest explants can be used for immunofluorescence. However, other techniques requiring more material are very difficult to carry out with these explants, because of the small amount of material obtained. The neural crest cell line Joma can also be useful for many applications. To study Schwann cell differentiation, dorsal root ganglion explants can be used to study the interaction between axons and Schwann cells. With these explants that contain sensory neurons and satellite glial cells (that differentiate into Schwann cells in culture), it is possible to produce "real" myelin in vitro. And when a pure Schwann cell population and a lot of material are needed, cultures of primary rat Schwann cells are the method of choice. To study protein/protein interactions, we commonly use the technique of coimmunoprecipitation, at least when we have an idea of the proteins that could interact together. Another possibility when to identify protein-binding partners is to analyze the protein content after immunoprecipitation by mass spectrometry. To study protein/dna interactions, we use chromatin immunoprecipitation (ChIP) analysis. This technique also requires that we have an idea of the region our protein of interest binds to. A more systematic approach is to couple ChIP to deep sequencing. This is a very powerful technique since it is supposed to identify all genes and regions of these genes a protein binds to. However, analysis of the data is still quite challenging, and only experienced bioinformatics analyzers can run this analysis properly. Microarray analysis is another very useful systematic approach for gene expression profiling between two biological samples. Analysis of the data is quite straightforward and provides lots of information at the mrna level. To determine whether your protein of interest activates or represses the transcription of a gene, luciferase gene reporter assay is very useful and a rather easy technique. To manipulate the expression of a protein, we can either use an shrna to downregulate the expression of this protein or in contrast we can overexpress this protein by cdna. To deliver an shrna or a cdna, cells are either transfected with a circular plasmid or transduced with a virus (lentivirus, adenovirus). In general, neural cells are more efficiently transduced with viruses than transfected with plasmids. As mentioned before, cleaved caspase 3 immunofluorescence or TUNEL assay are used to detect apoptotic cells. Figure 10 illustrates the techniques of choice for mechanistic analyses. 10

11 1. Protein/Protein interactions: Co-immunoprecipitation 3. Transcriptional activity due to protein binding : Luciferase gene reporter assay 5. Cell survival/ apoptosis: Cleaved caspase 3 immunofluorescence or TUNEL assay 2. Protein/DNA interactions: Chromatin immunoprecipitation Figure 10. Techniques to use for mechanistic analyses 4. Regulation of protein expression: Downregulation by shrna or Overexpression, followed by Western blot or Immunofluorescence or RT-PCR 10.7 Conclusion The development of Schwann cells and their cell cycle during regeneration after a lesion are tightly controlled by a network of transcription factors that are themselves regulated by chromatin remodeling enzymes such as HDACs. Other chromatin remodeling enzymes such as histone methyltransferases (HMTs) and demethylases (HDMs) are also crucial for the development of the nervous system, but we do not have the time in this lecture to develop this topic. Just keep in mind that HDACs always act within multiprotein complexes, and these complexes often contain also HMTs or HDMs, showing that there is cooperation of different chromatin remodeling enzymes to fine-tune the expression of target genes. For information (only), Figure 11 summarizes the know functions of HDACs in Schwann cells and oligodendrocytes (myelinating glial cells of the CNS). Although Schwann cells and oligodendrocytes share the same function of myelinating axons, their differentiation process is not controlled the same way, and HDACs act through different mechanisms in these two cell types. This illustrates the fact that HDACs have different functions and mechanisms of action, depending on the cell type. This could be explained by different patterns of protein expression depending on the cell type and therefore by the formation of different protein complexes. 11

12 Figure 11. Known functions of HDACs in the development of Schwann cells (a) and oligodendrocytes (b). 12