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Home , Epigenetics, Genetics, Gene expression, Molecular genetics

CHAPTER 21 , Stem Cells, , and Associated Hereditary Neurological Disorders Bhairavi Srinageshwar*, Panchanan Maiti*,**, Gary L. Dunbar*,**, Julien Rossignol* *Central Michigan University, Mt. Pleasant, MI, United States; **Field Institute, Saginaw, MI, United States

OUTLINE

Introduction to Epigenetics 323 and Their 325 Epigenetics and Neurological Disorders 326 Epigenetics and the 324 Stem Cells 324 Conclusions 335 Eukaryotic Chromosomal Organization 325 References 336

INTRODUCTION TO EPIGENETICS changes are discussed in detail elsewhere [3,4], but are briefly described later as an overview for this chapter. Epigenetics is defined as structural and functional DNA . DNA methylation and some of the changes occurring in histones and DNA, in the absence modifications are interdependent and play an of alterations of the DNA sequence, which, in turn, has important role in activation and repression during a significant impact on how is altered [5]. DNA methylation reactions are cata- in a [1]. The term “epigenetics” was coined by the lyzed by a family of called DNA methyl trans- famous developmental , Cornard Hal Wad- ferases (DNMTs), which add methyl groups to a dington, as “the branch of that studies the causal base of the DNA at the 5’-end, giving rise to the 5’-methyl interactions between and their products, which cytosine. This reaction can either activate or repress gene bring the into being”[2]. Epigenetics bridge expression, depending on the site of methylation and it the gap between the environment and gene expression, can also determine how well the enzymes for gene tran- which was once believed to independently [3]. scription can access the DNA that is wrapped around the Epigenetic changes can lead to increase or decrease in histone [6]. gene expression, thereby activating and/or deactivating . Trimethylation of at position genes, depending on the of the epigenetic con- 4 on histone 3 () promotes gene trol. Some of the most important histone modifications (i.e., gene activation), whereas trimethylation of lysine include: (1) methylation; (2) ; (3) phosphory- at position 27 on histone 3 () inhibits gene lation; (4) ubiquitination; and (5) SUMOylation and transcription (i.e., ). Alternate gene acti- DNA modification, including DNA methylation. These vation and repression promote a balanced dose of gene

Handbook of Epigenetics. http://dx.doi.org/10.1016/B978-0-12-805388-1.00021-3 Copyright © 2017 Elsevier Inc. All rights reserved. 323 324 21. Epigenetics, Stem Cells, Cellular Differentiation, and Associated Hereditary Neurological Disorders expression, which is required for those genes involved in The outer layer of the , called ectoderm, forms overall development and maturation of . This the central nervous system, and during the process of ensures that only appropriate genes are turned “ON” development, DNA methylation controls the epigen- and “OFF” at any given point of time [7]. Huntington’s etic mechanisms of the embryonic cells. For example, (HD), Parkinson’s disease (PD), and multiple to prevent the differentiation of nonneuronal cells into sclerosis (MS) are some of the that are caused by mature , the proneural genes, as well as gene abnormal DNA methylation pattern. These are discussed which are associated with involved with neu- in more detail in later sections of this chapter. rogenesis, such as the brain-derived neurotrophic factor Histone acetylation. DNA acetylation involves the (BDNF) gene, are silenced by DNA methylation at their acetylation of lysine residue, which is catalyzed by the region. However, DNA remethylation can enzymes, histone acetyltransferases (HATs), and histone take place on a neuronal gene, such as [11], which deacetylases (HDACs), which have opposing effects on allows for the selective initiation of neuronal develop- each other. HATs transfer acetyl groups to lysine, whereas ment. Similarly, during initial development of the cor- HDACs remove acetyl groups from lysine. However, tex, the genes involved in the formation of glial cells are presence or absence of an acetyl group (CH3CO) on a suppressed by DNA methylation, promoting the forma- lysine residue alters the charge on the and tion of more neurons during the early stages of neuro- can decrease the interaction of the N-terminal region of nal development. Eventually, during the later stages of histones with the negatively charged phosphate groups cortical development, the DNA methylation is reversed, of DNA. These events are involved in transformation of leading to the generation of glial cells [12,13]. Some of the condensed into a more relaxed structure, the genes involved in postnatal are Sox2, which can induce gene expression [8]. Dlx2, Sp8, and Neurog2 and those involved in the for- Histone . Phosphorylation involves addi- mation of glial cells are Sparcl1 and Nkx2-2. It has been tion of phosphate group to threonine, serine, and tyrosine shown that during the differentiation of neural stem residues. Phosphorylation of serine at position 139 on cells (NSCs) or progenitor cells, DNA methylation is histone 2 (H2) occurs as a response to DNA damage dur- facilitated by DNMT3a, which silences the genes Sparcl1 ing cell cycling. This relaxes the chromatin; thereby the and Nkx2-2, leading to the inhibition of glial cell for- proteins or factors responsible for repairing the damaged mation and promotion of mature development regions of the DNA have greater access to the DNA, which from NSCs [14]. It is also believed that the normal aging aids in the recovery of the DNA damage. Moreover, phos- process in is associated with modification of the phorylation of threonine and serine residues on histone 3 in the brain, affecting certain genes related (H3) facilitates regulation of gene expression [9]. Histidine to neurogenesis, especially within the cortex [15]. This phosphorylation is one of the epigenetic modifications process leads to the disruption of synapses, abnormal occurring in the prokaryotic cells and in lower neurotransmission, and is associated to age-related dis- that plays a major role in . The histidine is orders [16]. However, a comprehensive description of phosphorylated at the imidazole ring, but occurs only on the epigenetic mechanisms related to aging is beyond those nitrogen that are unprotonated [10]. the scope of this chapter. Histone ubiquitination and SUMOylation. Ubiquitina- tion and SUMOylation are associated with posttransla- tional modifications that regulate transcription of gene Stem Cells and activities. It is well established Dysregulation of epigenetic mechanisms has a direct that the addition of to proteins facil- impact on gene expression patterns that lead to abnor- itate targeted protein degradation. In addition to ubiqui- mal gene functions, which form the basis of most of the tin, various small ubiquitin-like molecules (SUMO) are genetic disorders (monogenic or polygenic) in humans. observed in cells, known as small ubiquitin-related mod- Environmental stress can also alter epigenetic mecha- ifier. These molecules have activities, which are similar nisms, which could become a cause for the predisposi- to ubiquitin and can attach covalently to those proteins, tion of certain diseases, such as , , which are involved in changing chromatin structure and and congenital heart disease [17]. This chapter focuses gene expression [3]. on the role of epigenetics and epigenetic changes that take place during differentiation, which can be used as a potential for neurological diseases. EPIGENETICS AND THE Stem cells have a unique property of proliferation, dif- ferentiation, and self-renewal. Stem cell plasticity is an Generation of neurons and glial cells from progeni- important characteristic and is based on the degree of tor cells involve epigenetic mechanisms that take place pluripotency, which is the ability of a cell to differenti- throughout the developmental stages of the brain. ate into another cell lineage. Stem cells can be classified

V. Factors Influencing Epigenetic Changes Epigenetics and the Human Brain 325

TABLE 21.1 An Overview of Various Epigenetic Mechanisms Associated With Neurodegenerative Diseases

Neurodegenerative disease Stem cell based therapy Epigenetic mechanism involved with disease

HD BM-MSCs Histone 3 (H3) methylation leading to reduced trophic factors [20]

PD NSCs DNA methylation leading to metabolic defects [21]

RTT iPSCs Point in MeCP2 gene leading to defective epigenetic regulatory molecules [22]

SCA UC-MSCs Methylation and acetylation of histone leading to reduced RNA expression [23,24]

MS HSCs DNA methylation, histone acetylation and posttranscriptional modification by miRNA leading to compromised immune response [25–27]

HD, Huntington’s disease; MS, ; PD, Parkinson’s disease; RTT, Rett ; SCA, spinocerebellar ataxias. as: (1) totipotent, such as embryonic stem cells (ESCs) , and the general hierarchy in the chromo- of the morula, which can be differentiated into any cell somal organization, is needed, which is briefly reviewed , including placental cells; (2) pluripotent, such as in the next section. induced pluripotent stem cells (iPSCs), which can be dif- ferentiated into any , except for placental cells; and (3) multipotent, such as mesenchymal stem cells Eukaryotic Chromosomal Organization (MSCs) and neural stem cells (NSCs), which can be dif- The strands of DNA are about 2 nm wide and are ferentiated into many, but not all, cell types. packed around the core of four pairs of histone pro- ESCs are highly unspecialized and can form any teins (H2A, H2B, H3, and H4), forming the , type of specialized cells under appropriate conditions which is the first level of chromosomal organization and environments. ESCs divide and renew themselves, (Fig. 21.1). These are the building blocks of which make them appropriate candidates for regenera- the chromatin structure, which is about 30 nm in diam- tive or cell replacement therapy [18]. Previ- eter. The formation of chromatin structure involves the ously, we have reviewed the role of epigenetics in MSCs, fifth histone, H1, which is near the adjacent nucleosome, NSCs, and iPSCs and their association with neurode- thereby compacting the nucleosome or chromatin to generative diseases [19]. In addition, the basics of stem form chromatin coils, which are about 300 nm in diam- cells, and the various epigenetic mechanisms associated eter. These fibers are further condensed to make loops with them, are explained comprehensively in the previ- of 700 nm in diameter, which, in turn, form the intact ous edition of this book. Therefore, the aim of the pres- metaphase chromosomes, which are about 1400 nm ent chapter is to discuss the role that epigenetics of stem wide [28]. cells might play in a subset of neurological diseases. Some of the inherited genetic disorders that occur as a consequence of abnormal epigenetic mechanisms Histones and Their Structure include: (1) HD; (2) PD; (3) (RTT); (4) spinocerebellar ataxia (SCA); and (5) MS. Currently, The consists of two molecules of each his- stem cell–based are being tested as a potential tone protein (H2A, H2B, H3, and H4) giving rise to an treatment for these diseases. A sampling of these treat- octamer, which is made of about 130 amino acids. The ment strategies are outlined in this chapter and include nucleosome, as discussed before, consists of DNA, that examples of: (1) transplants of bone marrow-derived is, 146–147 long, making about 1.65 turns mesenchymal stem cells (BM-MSCs) for treating HD; around the octamer. The core histones are highly con- (2) transplants of neural stem cells (NSCs) for treating served in eukaryotes, having a “tail” at their N-terminal PD; (3) transplants of induced pluripotent stem cells end where the epigenetic modifications, such as meth- (iPSCs) for treating RTT; (4) transplants of umbilical ylation, acetylation, and/or phosphorylation, take cord-derived mesenchymal stem cells (UC-MSCs) for place. These regulate the chromatin structure, which treating SCA; and (5) transplants of hematopoietic stem has an impact on recruiting various proteins involving cells (HSCs) for treating MS. These treatment strategies activation and repression of gene expression [28,29]. and the epigenetic mechanisms involved with these dis- Defects in chromatin organization and deficiency of orders are summarized in Table 21.1. enzymes lead to various forms of human diseases, such To better understand the role of epigenetics in neuro- as RTT, Rubinstein–Taybi syndrome, and Coffin–Lowry logical disorders, a working knowledge about histones, syndrome [30].

V. Factors Influencing Epigenetic Changes 326 21. Epigenetics, Stem Cells, Cellular Differentiation, and Associated Hereditary Neurological Disorders

FIGURE 21.1 The eukaryotic chromosomal organization. The steps showing the chromosomal organization involved in condensation of a 2 nm wide DNA wrapping around the histone molecules to form the nucleosome, which in turn twists to form coils (30 nm) and loops (300 nm) which further condenses into a 1400 nm wide [28].

Epigenetics and Neurological Disorders Currently our laboratory is investigating the role of transplanting BM-MSCs and UC-MSCs as a potential Huntington’s Disease and Mesenchymal Stem Cells treatment for HD. We have shown that BM-MSCs cre- Huntington’s disease (OMIM #143100) is a devastat- ate an optimal microenvironment in the that ing, fatal, autosomal dominant neurodegenerative dis- slows the progression of neuronal loss and dysfunction order, which most profoundly affects the striatal region by restoring the various neurotrophic factors, includ- of the brain. The disease is characterized by cognitive, ing BDNF, which is down regulated in HD. In our pre- motor, and psychiatric disturbances [31]. There is atro- vious studies, we transplanted BM-MSCs, which were phy and loss of the medium spiny GABAergic neurons genetically altered to overexpress BDNF, into the striata in the caudate and putamen regions of the striatum, of YAC128 mice (a slowly progressing, transgenic HD which leads to motor and cognitive impairment [32]. mouse model, which carries the entire human mHTT The disease is due to the expansion of CAG repeats in gene) and R6/2 mice, (fast-progressing, transgenic HD the gene (HTT), which produces mouse model, carrying 1 of human mHTT gene), huntingtin protein (mHTT) that is highly toxic, leading and observed profound neuroprotective effects, includ- to the of the disease [33]. ing a significant reduction in the motor symptoms of the

V. Factors Influencing Epigenetic Changes Epigenetics and the Human Brain 327 disease. These observations suggest that manipulation cofactors to regulate neuronal gene expression. REST, a of BM-MSCs could be a potential therapeutic for allevi- transcriptional , plays a major role in the regu- ating HD [34–36]. lation of the BDNF gene. Under normal circumstances, The Huntingtin gene. The normal HTT protein is local- REST binds with the HTT in the , but in the ized in the cytoplasm, whereas mHTT is found in the case of mHTT, there is no interaction between the REST nucleus, as well as in the cytoplasm. Though both wild and HTT (Fig. 21.2). This leads to nuclear transloca- type HTT and the mHTT can interact and inhibit acet- tion of the REST, which then inhibits BDNF expression, yltransferase function, the actual presence of the mHTT resulting in . REST not only targets in the nucleus was shown to be specifically responsible BDNF gene, but also influence other neuronal genes that for interfering with the acetylation of histones [37]. The are down regulated in HD [41]. cognitive symptoms observed in HD are linked to the These findings show that rescue of REST-regulated hypermethylated status of certain genes, such as Sox2 genes may prove to have a promising therapeutic effect and Pax6. Because these genes play vital roles in the pro- on HD. H3K4me3 levels are significantly lower at the liferation and maintenance of neural stem cells, which REST region of the BDNF gene in the cortex when com- eventually become neurons, alterations of their struc- parisons are made between 8 and 12-week old R6/2 mice. ture may contribute to subsequent dysfunctions, such H3K4me3 not only regulates BDNF gene expression, but as an impairment of hippocampal neurogenesis [38]. also has an impact on other genes, a postulate that was Similarly, the hypoacetylation of certain genes, such as confirmed by analysis and genome-wide BDNF gene, has been found in HD patients and analysis, which revealed that there are about 98 major models of HD. The CREB binding protein (also known genes showing differential expressions in the cortex and as CREBBP) is a histone acetyltransferase and a tran- the striatum in 12-week old R6/2 mice. In the cortex, the scriptional cofactor that regulates histone acetylation differentially expressed genes are associated with neuro- and gene activation. When the mHTT interacts with the transmission (e.g., Grla3, Grm4, and Bagra5), G-protein CREB binding protein, it loses its transcriptional activa- signaling (e.g., Rgs9 and Arpp21), synaptic transmis- tor and HAT functions, which lead to hypoacetylation sion (e.g., Snap25 and Rph3a), inflammation (e.g., of histones. This in turn, leads to transcriptional dys- and Dusp6), and calcium signaling (e.g., Scn4b, Hpca, regulation of certain genes in the neurons in HD brain and Itpr1). In the striatum, the differentially expressed [39]. Later, it was found that defects, other than DNA genes are associated with neurotransmission (e.g., Drd2, methylation and acetylation, are involved in HD. Lee Grm3, and Gabrd), G-protein signaling (e.g., Rgs9 and and coworkers [40] investigated the role of microRNA Arpp21), synaptic transmission (e.g., Snapr and Dlg4), molecules (miRNA) and found lower levels of 9 types of and calcium signaling (e.g., Scn4b, Hpca, and Itpr1) [20]. miRNA in 12-month-old YAC128 and 10-week-old R6/2 Stem cell therapy for HD. MSCs are adult stem cells that mice. Understanding the defective epigenetic mecha- are abundantly present in bone-marrow (BM). MSCs can nisms in HD has led to the use of HDAC inhibitors and also be derived from umbilical cord (UC) and adipose miRNA as potential treatments for this disease. (AT). The MSCs, derived from BM and AT, have HD and BDNF. BDNF is an important neurotropic a greater survival rate, when compared to the MSCs factor, which is expressed abundantly in different brain derived from other sources [43]. BM-MSCs can differen- regions. H3K4me3 is a histone involved in transcription tiate into osteogenic, adipogenic, and chondrogenic lin- of the BDNF gene, which is significantly reduced in the eages. However, by triggering an epigenetic mechanism, cortex of the HD brain. BDNF is expressed in the corti- these MSCs can differentiate into a neuronal-like lineage. cal neurons that project into the striatum and has been shown to be essential for the survival of striatal neurons. As mentioned earlier, histone methylation leads to gene silencing and histone acetylation leads to gene activa- tion. However, H3K4 is an exception because either methylation or acetylation of this histone leads to gene activation. Modification of H3K4me3 is widely studied and is of interest to researchers because H3 is associ- ated with the promoters of the genes that are actively transcribed (e.g., BDNF) [41]. Research using chromatin immunoprecipitation (ChIP) analysis has revealed a cor- FIGURE 21.2 Role of REST in downregulation of BDNF in HD. relation between BDNF expression and H3K4me3 levels (A) The wild-type HTT protein binds to the REST protein in the cy- toplasm, thereby preventing the REST to bind to the BDNF in HD [20]. In the brain, repressor element-1 silencing promoter. (B) The mHTT fails to bind to the REST which causes REST /neuron-restrictive fac- to bind to BDNF promoter and inhibits trophic factor transcription tor (REST/NRSF) is the main factor that recruits other leading to reduced BDNF expression as seen in HD brain [42].

V. Factors Influencing Epigenetic Changes 328 21. Epigenetics, Stem Cells, Cellular Differentiation, and Associated Hereditary Neurological Disorders

This can be achieved by passaging the MSCs and every chromosomal abnormalities that may adversely affect time the MSCs get passaged, a series of methylation and survivability and successful engraftment at the trans- acetylation reactions take place [44]. plantation site [47]. Therefore, even though higher- Recently, we have found that MSCs at higher pas- passaged MSCs may have a stronger therapeutic effect sages (passaged about 40–50 times prior to transplan- for HD, it is important to study the epigenetic mecha- tation) have more therapeutic for alleviating nisms of MSCs at different passages to select a subpopu- motor symptoms in the R6/2 mice, compared to lower- lation of cells that will show their maximum therapeutic passaged MSCs (passaged about 3–8 times prior to effects, without adverse effects. transplantation). This study showed that passaging the Epigenetic mechanisms play an important role in cells up to 40–50 times produces a subpopulation of the dysregulation of genes that are associated with the MSCs that have the potential to create an optimal envi- of trophic factors, such as BDNF, as described ronment within the transplantation site in the striatum, previously. Therefore, targeting epigenetic markers as well as generating BDNF, which is usually deficient in and improving the expression of BDNF may prove to HD [36]. Genes that are associated with steering be beneficial in alleviating the signs and symptoms of the MSCs toward osteogenic, adipogenic, and chondro- HD [20]. genic lineages, such as (OPN), peroxisome Although various studies have described histone proliferator-activated receptors gamma 2 (PPAR-γ2), and modifications and histone variants that are associated fatty acid binding protein 4 (FABP4), undergo methyla- with HD, including phosphorylation of histone 2 vari- tion and histone acetylation, as the cells are passaged, ants observed in HD cell line and in R6/2 mouse model thereby reducing differentiation into osteogenic, adipo- [48], a detailed description of this and many other his- genic, or chondrogenic lineages [44,45]. The acetylation tone variations is beyond the scope of this chapter. status of H3K9 at the promoter regions of these genes undergoes changes, thereby leading to less activation Parkinson’s Disease and Neural Stem Cells [46]. Therefore, when higher passaged MSCs are trans- Parkinson’s disease (OMIM #168600) is a late-onset planted into animal models, the environment at the neurodegenerative disease, mainly affecting individuals transplantation site is more favorable for increasing of about 65–85 years of age. The disease is characterized BDNF, which is required for neuronal survival and alle- by impairment of both motor and nonmotor symptoms, viating symptoms of HD [36]. including rigidity, bradykinesia, tremor, postural insta- Similarly, in another study, we genetically modified bility, depression, abnormal sleep patterns, cognitive the BM-MSCs to overexpress BDNF and transplanted dysfunction, and autonomic insufficiency[21,49] . PD these cells into the striatum of the YAC128 mice. The is due to the degeneration of dopaminergic neurons in mice, which received BM-MSCs that overexpressed substantia nigra, pars compacta (SNpc). The genetic fac- BDNF showed improvement in motor coordination, tors, such as mutations in PARK genes and environmen- compared to YAC128 mice, which did not receive BM- tal factors, such as aging and exposure to neurotoxins, MSCs that overexpressed BDNF [34]. Interestingly, one contribute to the disease [50,51]. As such, PD can be of our previous studies involved transplantation of caused either by sporadic mutations or can be inher- UC-MSCs into the striata of the R6/2 mice and though ited. Major gene candidates that are associated with PD, these mice showed reduction in both spatial include PARK genes, leucine-rich repeat kinase 2 gene and motor deficits, the extent of behavioral sparing was (LRRK2), and the α-synuclein gene (SNCA). The muta- slightly higher when BM-MSCs were used, suggesting tions in PARK (PARK 1–15) and SNCA show that the source of the MSCs may affect their efficacy patterns, suggesting familial PD. when transplanted [35]. Sporadic PD is caused by variants found in SNCA and Conclusions for epigenetics in HD. The epigenetic altera- LRRK 2 genes. PD also shows polygenic and complex tions of genes, such as OPN, PPAR-γ2, and FABP4, steer inheritance patterns, combined with environmental MSCs away from osteogenic, adipogenic, and chondro- factors [21]. genic lineages as a function of cell passaging and may Impairment of one-carbon in PD. The group play an important role in driving these stem cells into a of metabolic reactions consisting of various enzymes neuronal-like lineage. The aforementioned studies show and coenzymes that are involved in various biological that MSCs have a therapeutic effect on HD and by uti- functions that involve metabolism, redox reaction, lizing higher-passaged MSCs, the transplants appear to and methylation reaction is known as one-carbon metab- be more efficacious than using lower-passaged MSCs. olism. These metabolic activities take place by utilizing However, the higher-passaged MSCs may have less glucose, amino acids, such as serine and glycine, and clinical utility, because they are more susceptible to vitamins, such as B12 and B6 [52]. Therefore, impairment other epigenetic effects and tumor formation follow- in DNA methylation is a part of one-carbon metabolism ing transplantation, as they have been shown to carry that is found in PD.

V. Factors Influencing Epigenetic Changes Epigenetics and the Human Brain 329

As mentioned earlier, DNA methylation is one of the and age-matched controls. DNA methylation analysis major epigenetic modifications that ensures condensa- revealed that there was no significant difference in meth- tion or relaxation of chromatin structure, depending ylation status between the three groups, which suggests on the methylation status of the DNA that is wrapped that PARK gene methylation does not contribute in the around the histones. The is catalyzed PD pathogenesis. by DNMT1, where cytosine gets methylated to form Stem cell therapy for PD. More than half of the dopami- 5′methyl-cytosine, a reaction that involves two major nergic neurons are lost in SNpc before the actual onset of molecules, S-adenosylmethionine (SAM), and S-adeno- PD [59]. There are various studies and literature reviews sylhomocysteine (SAH). SAM is a universal methylating that have investigated the role of neural stem cells in agent produced from folate and homocysteine (HCY), PD [49,54,60] and the importance of neurotrophic fac- which methylates histones and DNA [21]. SAM, SAH, tors, particularly glial cell line-derived neurotrophic fac- and HCY are some of the of the metabolic tor (GDNF). The NSCs are multipotent stem cells that pathway associated with the one-carbon metabolism are specifically found in the (SVZ), [53]. Hence, the DNA methylation potential depends on subgranular zone (SGZ), and the dentate gyrus (DG) the levels of SAM and the potential of DNMT1 to cata- of the . The environment at these regions lyze the reaction and transfer the methyl group to cyto- are the most favorable for the differentiation of NSCs sine. This reaction was found to be impaired in many into neurons [61]. Sanberg [60] has discussed the role of neurodegenerative diseases, especially in genes asso- transplantation of undifferentiated NSCs into the stria- ciated with Alzheimer’s disease (AD) and PD. Due to tum of a primate model of PD and indicated that NSCs defects in one-carbon metabolism, the rate of methyla- were able to survive and migrate to the site of neuro- tion decreases, leading to increased gene expression. An degeneration and replace the lost neurons. The example of such defects in metabolism was found dur- recovered from their behavioral deficits. Redmond and ing the analysis of methylation status of the SNCA gene coworkers [62] also transplanted undifferentiated NSCs in PD patients [21]. into the primate model of PD and found that though, The SNCA gene. The α-synuclein protein is important these undifferentiated NSCs had therapeutic effect, only for dopaminergic neurogenesis during early embryonic a small of these NSCs partially differentiated development. The Lewy bodies found in PD consist of into dopaminergic neurons, due to a less-than optimal SNCA and protein inclusion bodies, which lead to dis- microenvironment at the site of transplantation. These ease pathogenesis. Lower levels of SNCA are associated studies indicate that the partially differentiated NSCs with loss of dopaminergic neurons during embryonic migrate to the substantia nigra, through the nigrostriatal stage, whereas increased expression of SNCA may be pathway, following their unilateral transplantation into a risk factor or a threat during later stages [54]. The the striatum. However, these finding suggest that cell genetic abnormalities associated with SNCA that lead to replacement therapy provides only minimal neuropro- PD are the point mutations and copy number variants. tective effects. Previous studies have reported that the of the Other approaches, such as dopamine replacement SNCA genes found in dopaminergic neuron, results in therapy and deep brain stimulation (which decrease decreased methylation, leading to mono-allelic expres- tremor and rigidity), also failed to show neuroprotec- sion of the gene. However, mRNA levels from this single tive effects. GDNF is well known to increase surviv- exceed that of the control subjects having normal ability of dopaminergic neurons, and delivering GDNF biallelic expression. Methylation status of the intronic into the brain of PD animal models, such as MPTP regions (human 1 having 66 CpG sites) of the (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated SNCA gene was found to be similar to those reported rhesus monkey [63] and 6-OHDA (6-hydroxydopamine) in previous studies, especially in reference to the cortex injected [64,65], were shown to be neuroprotective. and substantia nigra regions of the brain [55,56]. The Overall, GDNF is critical for establishing the nigrostria- miRNA also plays a role in regulating the gene expres- tal dopamine system during development and plays an sion of SNCA. Doxakis [57] has analyzed two of the important role in protecting dopaminergic neurons from major miRNA of the brain, mi-RNA7 and mi-RNA153, degeneration by maintaining their morphology and and found that overexpression of these miRNAs in neu- neurochemical and biochemical reactions that are taking ronal culture lowered the SNCA levels. place in them, as well as ensuring proper neuronal dif- The PARK gene. The majority of PARK gene muta- ferentiation and long-term survivability of neurons [66]. tions are also associated with juvenile form of PD. Cai Open-labeled clinical trials using GDNF showed tol- and coworkers [58] investigated about 33 CpG regions erance and clinical benefits in patients within 3 months on the PARK gene promoter in three groups of indi- of treatment, but randomized clinical trials failed to viduals, including PD patients carrying PARK gene reveal significant benefits [67]. Deng and coworkers [68] mutations, PD patients without PARK gene mutations, have shown that the cotransplantation of dopaminergic

V. Factors Influencing Epigenetic Changes 330 21. Epigenetics, Stem Cells, Cellular Differentiation, and Associated Hereditary Neurological Disorders neurons and NSCs can reduce motor symptoms in a disorder affecting, predominantly, the popula- model of PD. However, to increase the number tion and classified as one of the autism-spectrum dis- of NSCs that differentiate into dopaminergic neurons, orders (ASDs). The classical symptoms of this disease the overexpression of nuclear (Nurr1) and fac- include speech disability, stereotypic hand use, autistic tors, derived from local type 1 astrocytes, are necessary. characteristics, and seizures that gradually develop after Wagner and coworkers [69] showed that NSCs having 18 months of age [22]. The cause of the disease is associ- these factors were able to differentiate into dopaminer- ated with point mutations in methyl-CpG binding protein 2 gic neurons, compared to NSCs that did not express gene (MeCP2), although there are other sets of genes and these factors. Similarly, Nurr1 and Pitx3 are needed environmental factors that contribute to the onset of this to produce dopaminergic neurons from embryonic disease. Most of the mutations observed in MeCP2 gene stem cells. Nurr1 is a nuclear receptor that is are point mutations (missense or nonsense). Some of the involved in the dopaminergic neurogenesis, while Pitx3 hotspots include: (1) p. R133C and p. T158M, found in is a transcription factor that is important for the differ- the methyl binding (MBD); and (2) p. R306C, p. entiation and maintenance of dopaminergic neurons in R168X, p. R294X, and p. R255X located in transcriptional the mid-brain. Nurr 1, along with Pitx3, influences the repression domain (TRD) [72–75]. The MeCP2 gene has expression of some of the genes involved in production a major role to play in the epigenetic regulation of vari- of dopamine, such as (TH) and ous gene expressions related to ASD [72]. The domains (DAT), which are associated with of MeCP2 gene, such as the MBD and TRD, are involved dopamine signaling [70]. Nurr1 is usually present in a in and protein interactions, silenced state when not combined with Pitx3. This is due respectively [76]. to the binding of silencing mediator of retinoic acid and MeCP2 gene and its function. MeCP2 gene is involved thyroid hormone receptor (SMRT) that leads to HDAC- in coding an epigenetic regulatory molecule, and muta- mediated silencing of Nurr1. However, in the presence tions or -scale deletions, duplications, and insertions of Pitx3, the binding of SMRT is reduced and Nurr1 is of MeCP2 cause RTT syndrome. RTT can be classified activated, thereby the Nurr1-Pitx3 complex can bind to into two categories, either atypical RTT or classical RTT. the promoter of TH and DAT genes and activate them. More than 95% of the patients having mutation in MeCP2 Therefore, Nurr1, on its own, cannot activate the target gene are considered having classical RTT. In general, genes associated with dopamine production [71]. MeCP2 binds to the DNA via the MBD and silences the Conclusions for epigenetics in PD. The familial form of gene. Similarly, the MeCP2 protein helps with chroma- PD involves defects in epigenetic mechanisms, such as tin remodeling by binding to the DNA via its TRD. The one-carbon metabolism reaction, which is associated methylation of histone 3 at lysine position 9 (H3K9me) is with defects in rate of DNA methylation that leads to achieved by MeCP2, thereby silencing the gene to which PD. The rate of transfer of methyl group to the cytosine it is bound [22]. However, Yasui and coworkers [77] by DNMT1 has been highly reduced, thereby interfering have extensively studied the MeCP2 binding sites on the with the one-carbon metabolism, which was confirmed genes and found that only about 6% of the CpG islands by measuring SAM and SAH, the biomarkers of the are bound by MeCP2. Their study indicated that: (1) the one-carbon metabolism associated with DNA methyla- main function of MeCP2 is not associated with silenc- tion. Similarly, abnormal increase in expression of cer- ing the methylated regions of the gene and (2) the genes tain miRNAs leads to decreased SNCA gene expression, having the maximum methylation status are not bound which is also associated with this disease. Cell replace- by MeCP2. Previous publications have shown that the ment therapy to increase production of GDNF through has methylated cytosine as 5-hydroxy- transplantation of partially differentiated NSCs has methylcytosine (5hmC) and 5-methylcytosine (5mC). It proven, thus far, to have only limited efficacy. To achieve has been shown that 5hmC is found abundantly in neu- conversion of NSCs into dopaminergic neurons, the ronal genes that are active and that MeCP2 has a very expression of Nurr1 and Pitx3 are required. The com- high affinity toward 5hmC compared to 5mC, which plex formed between Nurr1 and Pitx3 is associated with plays a major role in how the gene expressions are regu- increased gene expression of TH and DAT, which, in lated in neurons (Fig. 21.3). turn, increases the production of dopamine. Therefore, An interesting finding is that the MeCP2 competes complexing Nurr1 and Pitx3 is necessary, since Nurr1, with the histone, H1, to bind to the nucleosome, indi- per se, is considered to be in a silenced state, and binding cating that the levels of H1 and MeCP2 are not always with Pitx3 leads to the activation of the complex. corelated with each other, especially in neurons. The finding that MeCP2 gene is associated with activating Rett Syndrome and Induced Pluripotent Stem Cells genes when bound to 5hmC, as well as with silencing the Rett syndrome (OMIM #312750) is a X-linked auto- genes when linked with H3K9, underlies the dual nature somal dominant progressive neurodevelopmental of the protein. Therefore, there are some genes that are

V. Factors Influencing Epigenetic Changes Epigenetics and the Human Brain 331

FIGURE 21.3 Function of MeCP2 gene. MeCP2 regulates the transcription of certain genes in the brain by binding to 5-methylcytosine (5mC). The main function of MeCP2 gene is not just silencing the gene to which it is bound, but also has the ability to increase the transcription of genes, such as BDNF, ORPM1, and CREB 1 [15]. down regulated when MeCP2 is lost and upregulated status, thereby emphasizing the dual role of the gene. with increased MeCP2 gene expression [78]. Although both RTT syndrome and iPSCs have well- iPSC models of RTT. Induced pluripotent stem cells known and strong epigenetic components, the use of have been used as a cell model for RTT [79]. Takahashi iPSCs for treating RTT has not been investigated. The and Yamanaka [80] first reprogrammed the cells stem-cell-based model of RTT is very useful to study into iPSCs by overexpressing four major genes, such as a specific mutation leading to a phenotype- Oct3/4, Sox2, , and c-Myc, which are now collectively correlation and correcting the mutated RTT-iPSCs, in known as the Yamanaka factors. of vitro, and then transplanting them may prove to be iPSCs involves loss and gain of DNA methylation on H3 a future treatment for RTT. Given that highly specific at lysine positions 27 (H3K27me3) and 4 (H3K4me3), as and targeted cell replacement therapy can be achieved the cells get transformed from somatic stage, to pluripo- using the corrected iPSCs, utilizing epigenetically cor- tent stage as has been discussed in detail in our previ- rected iPSCs for treating RTT syndrome is worthy of ously work [19]. further investigation as such an approach has signifi- The iPSCs derived from the RTT patients have been cant promise. reprogrammed to form neurons, which show signifi- cant pathological changes, such as reduced nuclear size, The Spinocerebellar Ataxia and lower expression of neuronal markers, reduced den- Mesenchymal Stem Cells dritic spine density, loss of synapses, and lower levels SCAs are a group of neurodegenerative disorders of intracellular calcium. Electrophysiological analysis that are caused by trinucleotide repeat expansions of neurons obtained from the RTT-derived iPSCs show (mainly CAG expansions that lead to elongated poly- decreased excitatory and inhibitory postsynaptic poten- glutamine tracts). There are about 30 different genes tial (EPSP and IPSP). Although cell replacement research responsible for the disease that is inherited in an auto- for RTT using iPSC transplantation has not been trans- somal dominant pattern. SCA patients have neuronal lated to the clinic, the in vitro remodeling of fibroblasts degeneration in cerebellum, brain stem, and spinal derived from the RTT patients to form iPSCs have been cord. The main characteristics of this disease are related successfully achieved by Marchetto and coworkers to retinopathy, neuropathy, cognitive dysfunction, and [81]. These findings are paving the way toward a bet- dementia [83]. Unfortunately, there is no cure for SCAs. ter understanding of the of the disease Approximately 28 different types of SCAs have been and for identifying drugs or treatments that are patient- discovered so far, with the most common forms being or mutation-specific[81,82] . SCA1, SCA2, SCA3, and SCA7. A detailed description Conclusions for epigenetics in RTT. In general, it has of the different types of SCAs is discussed by Paulson been assumed that MeCP2 usually silences the gene [84]. Interestingly, SCA1, SCA2, SCA3, SCA6, SCA7, to which it is associated, but subsequent studies have and SCA17 are due to the repeat expansion in the cod- indicated that this might not always be the case. ing regions of the gene, whereas SCA8, SCA10, SCA12, The MeCP2 gene can increase, as well as decrease, and SCA31 have repeat expansions in the noncoding the gene expression depending on the methylation regions of the gene [84].

V. Factors Influencing Epigenetic Changes 332 21. Epigenetics, Stem Cells, Cellular Differentiation, and Associated Hereditary Neurological Disorders

SCA1 (OMIM #164400); and coworkers [88] showed that UC-MSCs provided a SCA1 mainly affects the brain stem and Purkinje cells therapeutic effect on these animals, including the resto- of cerebellum and is characterized by ataxia of limbs ration of motor functions at 8 weeks posttransplantation. and abnormal gait, leading to chorea. Assessments of The results of this study also showed that the cerebellar expanded Ataxin1 gene having 82 CAG repeats in the atrophy and the number of cells undergoing cerebellar Purkinje cells in SCA1 animal models have were reduced. There was also an increased production shown reduced gene expression, which is involved of growth factors, such as -like -1 in signal and calcium [85]. (IGF-1) and vascular endothelial growth factor (VEGF). Ataxin 1 interacts with two major proteins, the retinoid Jin and coworkers [89] performed intravenous and intra- acid receptor-related orphan receptor α (RORA, a type thecal transplantation of UC-MSCs into patients affected of HAT), and acetyltransferase tat-interactive protein 60 with SCA and found that UC-MSCs are safe and have (Tip60, MW 60kDa), which is a coactiva- the capacity to alleviate the symptoms in SCA patients. tor that helps in the interaction of RORA [using ATXN1 Another clinical study, conducted by Dongmei and with HMG-box protein 1 (AXH) domain of ATXN1]. coworkers [90] showed that transplantation of UC-MSCs However, in the presence of the mutant Ataxin 1 gene could alleviate the symptoms of SCA, without any side or protein, the interactions with Tip60 and RORAα are effects, when evaluated using International Cooperative disrupted, which lead to disease pathogenesis [86]. Ataxia Rating Scale (ICARS) and Activity of Daily Liv- ing Scale (ADL). Most importantly, these clinical find- SCA 7 (OMIM # 164500); ings provide converging evidence with the preclinical The symptoms associated with SCA 7 include cerebel- experimental results that UC-MSCs are safe and capable lar neurodegeneration and retinal degeneration. Similar of alleviating the symptoms of SCA, indicating a poten- to SCA 1, SCA7 also interacts with transcription factor tially safe and efficacious therapy for SCA. cone-rod protein (CRX). CRX is a transcrip- Conclusions for epigenetics in SCA. There is a strong cor- tion of genes that are involved in formation of relation between epigenetic defects in the genes Ataxin photoreceptors in the eyes. The interactions of mutant 1, Ataxin 7, and Ataxin 8 and the neuropathological phe- SCA7 with CRX cause abnormal formation of the pho- notypes associated with SCA in patients. Each ataxin toreceptors, leading to retinal degeneration. Although type has its own associated epigenetic mechanism. For the actual function of Ataxin 7 gene is unknown, it has example, the Ataxin 1 is a complex that is associated recently been shown that Ataxin 7 is a subunit of HATs with RORA and Tip60, which are acetyltransferases and [23]. Therefore, the mutant form of the protein is involved the interaction with them is disrupted in the presence of in the alteration of HATs, which eventually leads to the mutant Ataxin 1. Similarly, Ataxin 7 is a part of HAT and disruption of histone acetylation. presence of mutant Ataxin 7 leads to improper histone acetylation. Detailed analysis of SCA 8 cell line revealed SCA 8 (OMIM # 608768); hyper- and hypomethylation of H3K9 and H3K14, SCA 8 is due to the combined repeat expansion of respectively. [24] CTG and CAG in the Ataxin 8 gene. Chen and coworkers To the best of our knowledge, there are no epigenetic [24] studied the SCA 8 cell line, or transcript known as mechanisms related to the UC-MSCs that would drive ATXN8OS. Epigenetic analysis of this transcript revealed the cells to take on a specific neuronal phenotype that increased levels of and hypoacetylation of would confer significant therapeutic effects. However, H3K14 that led to repression of ATXN8OS RNA in cell previous studies have shown a favorable safety profile lines that have 157 repeats. Similarly, methylation of argi- and beneficial therapeutic effects of UC-MSCs for SCA. nine residues and phosphorylation of serine or threonine Multiple Sclerosis and Hematopoietic Stem Cells were found in the cell lines with about 88 repeats, which eventually led to decreased RNA expression. Multiple sclerosis (OMIM #126200) is an autoim- Stem cell therapy for SCA. Although preclinical and mune neurological disease characterized by the loss of clinical trials have been conducted using drugs, anti- myelin sheath, leading to demyelination and neurode- oxidants, and neurotrophic factors, none of these tri- generation of brain and spinal cord. The majority of the als were successful in alleviating the symptoms in SCA affected individuals are between 20 and 40 years patients [87]. However, UC-MSCs transplantations in a of age. In MS, the T-lymphocytes become stimulated by mouse model of SCA produced promising effects. The various factors, which, in turn, activate the inflamma- major advantages of using UC-MSCs are: (1) there are no tory pathways, leading to the symptoms of the disease ethical issues that arise from their use; (2) they are highly [91]. MS involves the genetic, epigenetic, and environ- multipotent stem cells; and (3) they have immunosup- mental factors (nutritional status). Epigenetic causes pressive properties, resulting in reduced risk of tumor include: (1) DNA methylation; (2) posttranscriptional formation posttransplantation. Using SCA mice, Zhang modification by miRNA; and (3) histone acetylation,

V. Factors Influencing Epigenetic Changes Epigenetics and the Human Brain 333 such as what occurs with the HLA-DRB 1 (human leu- kocyte antigen having beta chain) gene on chromosome 6, which is responsible for the production of major his- tocompatibility complex class II (MHC class-II) antigen and which plays an important role in immune response mechanisms [25,92]. The environmental factors include deficiency and frequent smoking which, in turn, leads to epigenetic changes observed in MS. DNA methylation in MS. Baranzini and coworkers [93] were the first to study the RNA transcriptome sequences and the epigenome sequences of CD4+ T-lymphocytes from three sets of MS-discordant, monozygotic , and found that there were no significant differences in the DNA methylation patterns. However, based on this study, alterations in DNA methylation patterns, as a cause of the disease cannot be ruled out, because the sample size was too small to make definitive conclu- sions. Other studies have shown that DNA methylation could be the cause of MS [94]. The methylation of CpG islands in some of the genes may be responsible for the disease, because the methyla- tion pattern determines how the two different types of T-helper-cells (Th1 and Th2) are formed, which, in turn, gives rise to cytokines, such as interferon-gamma (IFN- FIGURE 21.4 Impact of DNA methylation on T-cells. Compro- γ), interleukin-2 (IL-2), interleukin-4 (IL-4), and tumor mised immune response exerted by Th1 and Th2 cells ( of necrosis factor-α (TNF-α). In MS, there is a dominance IFN-γ over IL-4) is due to DNA methylation that leads to MS. of IFN-γ expression associated with Th1, compared to IL-4 molecules that are associated with Th2. There- directly associated with the initiation of the disease. It fore, abnormal DNA methylation pattern that is found was found that the patients have more severe form of in the promoter region of IFN-γ may explain why the disease; following relapse, and showed a high expres- immune response by Th1 is greater than Th2, leading to sion of mi-RNA326 (Table 21.2). This, in turn, leads to the pathophysiology of the disease (Fig. 21.4). Similarly, higher activation of T-cells and increased expression of DNA methylation or histone deacetylation is associated inflammatory cytokines, which then results in abnormal with the IL-4 genes, leading to the gene silencing. HDAC immune response, leading to MS [26]. inhibitors are given as a potential treatment for MS, Histone Acetylation. In 2007, the International Multiple because they lead to reduction in inflammation, demy- Sclerosis Genetics Consortium (IMSGC) conducted a elination, and neuronal degeneration [95]. genome-wide association study (GWAS) that included Epigenetics not only control cytokine gene activation about 12,000 subjects. It was found that two genes, other in MS, but also affects myelin structure, by regulating than HLA-DRB 1, are associated with MS. Single nucleo- (MBP). For example, hypomethyl- tide polymorphisms (SNPs) in interleukin-2 receptor α ation of an , called peptidyl-arginine deiminase gene (IL2RA) and interleukin-7 receptor α gene (IL7RA) 2 (PAD 2), has been observed in MS [96]. This enzyme is were found to be risk factors for MS. Again, in 2011, responsible for conversion of arginine to citrullin and its IMSGC conducted another GWAS study and found that level is increased in MS. Due to overactivation of PAD abnormalities in some of the genes involved in cytokines 2, the MBP is citrullinated and becomes vulnerable for and pathways cause MS, reinforcing degradation by the myelin-associated proteases, such the fact that the most commonly affected gene by histone as . This further leads to a reduced binding acetylation was the HLA-DRB 1 gene [25]. capacity of the MBP, causing lipid vesicle fragmentation, Stem cell therapy for MS. Among different stem cell pop- which, in turn, leads to the myelin breakdown observed ulations, HSCs have drawn a special attention for use as in MS [96] (Fig. 21.5). a potential therapy of MS, because of their multipotency. Micro-RNA. In vitro analysis of MS induced lesions These stem cells differentiate into a very large popula- and pooled cells have shown that there were about ten tion of cells, including all functional types of blood cells, miRNAs that were upregulated, especially mi-RNA155 B-cells, T-cells, and many other cell types, which make and mi-RNA326. However, mi-RNA326 is more com- them a promising candidate for therapy of many dis- monly associated with disease relapse, rather than being eases [97]. The first therapy trials using HSCs was started

V. Factors Influencing Epigenetic Changes 334 21. Epigenetics, Stem Cells, Cellular Differentiation, and Associated Hereditary Neurological Disorders

FIGURE 21.5 Comparison of citrullinated myelin in normal and MS patients. Hypomethylation causes increase in the production of enzyme peptidyl-arginine deiminase 2 (PAD 2) leading to increased citrullinated myelin basic protein, thus gives abnormal structure and thereby myelin breakdown in MS patients [92].

TABLE 21.2 Roles of Different Types of miRNA in MS miRNA Role in MS mi-RNA155 Dysregulation of gene expression in CD4+ cells and peripheral blood mononuclear cells [27] mi-RNA326 Dysregulation of gene expression in CD4+ [27] mi-RNA18b, mi-RNA493, and mi-RNA599 Increased expression in relapse remitting MS [27] in 1995, followed by a second successful therapy trial on they are extracted from bone-marrow and transplanted humans in 1998. These studies revealed that scores on into MS patients, the B-cells, and, especially the T-cells, the Expanded Disability Status Scale (EDSS) improved mature and become activated, which play a major role for patients who received the HSCs, confirming their use in boosting the immune levels. However, because it is as a potential treatment for this disease [98,99]. However, important to ensure that other immune cells, such as subsequent clinical trials [100] revealed that patients macrophages, do not populate the regions near the graft, who have a severe form of this disease (based on their it is advisable to use immune ablative conditioning regi- EDSS score) and who received the transplants did not mens to achieve the maximum benefit from the T-cells. show significant benefits from the cell treatment. It is A phase II conducted by Mancardi and possible that these stem cells may exert their beneficial coworkers [102] was reported recently in 2015, which effects only during the early stages of disease and not showed that autologous HSC transplantation resulted after the disease has progressed to its severe stage [100]. in suppression of lesion-induced inflammation. These Most of the transplants associated with the HSCs are results are based on a 4-year follow up of patients having autogenic in nature, because allogeneic or HLA-matched a progressive or a relapsing form of the disease. transplants cause an increase in mortality [91]. Collectively, these studies show that HSCs, due to Atkins and Freedman [101] reviewed the pros and their multipotent and immunomodulatory properties, cons of using HSCs as a potential treatment for MS. have a significant promise for producing an effective One of the major advantages of using HSCs is that once therapy for MS.

V. Factors Influencing Epigenetic Changes Conclusions 335

Conclusions for epigenetics in MS. The compromised dysregulated epigenetic markers in each disease (such immune response seen in MS is due to the epigenetic as H3K4me3 in HD and H3K9 and H3K14 in SCA8) mechanisms that affect the T-cells and the cytokines, may lead to designing a more targeted therapy for which form the major molecules or proteins of the such neurological disorders. Addressing these types of immune system. Hypomethylation of PAD 2 enzyme issues and furthering our knowledge about the epigen- is observed in patients affected with MS, which leads etic mechanisms of stem cells and the diseases associ- to the breakdown of the MBP. Further analysis showed ated with epigenetic alterations could pave way toward that miRNA, such as mi-RNA155, mi-RNA326, mi- developing more effective and long-lasting therapeutic RNA18b, mi-RNA493, and mi-RNA599 are associated approaches. with MS. The GWAS study and other studies have shown that HLA-DRB 1 is the main for MS. However, there are some SNPs associated other Abbreviations genes, such as IL2RA and IL7RA, that pose a risk factor ADL Activity of Daily Living Scale for this disease. HSCs have been used as a stem-cell- ASDs Autism-spectrum disorders based therapy for MS, whereby their activation of the AT T-cells improve immune responses, producing favor- BDNF Brain derived neurotrophic factor BM Bone marrow able outcomes. BM-MSCs Bone marrow-derived MSCs cAMP Cyclic adenosine monophosphate CBP CREB-binding protein CONCLUSIONS ChIP Chromatin immunoprecipitation CRE cAMP response element CREB CRE-binding protein Epigenetics not only play a role in determining CRX Cone-rod homeobox protein the stem cell fate, but also form the underlying bases DAT Dopamine transporter of neurodegenerative diseases, such as HD, PD, RTT, DNMTs DNA methyl transferases SCA, and MS, as discussed in this chapter. Although EDSS Expanded Disability Status Scale the genetic bases of these diseases vary, the epigenetic EPSP Excitatory post-synaptic potential ESCS Embryonic stem cells causes are similar. For example, alterations in the levels GDNF Glial derived neurotrophic factor of DNA methylation and histone acetylation gives rise GWAS Genome-wide association study to SCA and MS. Understanding the epigenetic mecha- HATs Histone acetyltransferases nisms of stem cells is an important aspect that needs HD Huntington’s disease to be carefully considered when designing strategies HDACs Histone deacetylases HLA-DRB Human leukocyte antigen having beta chain to achieve optimal efficacy and efficiency when cell HSCs Hematopoietic stem cells HSCs replacement therapy for neurodegenerative diseases is 6-OHDA 6-Hydroxydopamine being considered. 5hmC 5-Hydroxymethylcytosine In order to translate and improve the outcomes of ICARS International Cooperative Ataxia Rating Scale clinical trials, further research on how epigenetics drive IFN α Interferon α IGF-1 Insulin-like growth factor-1 stem cell fate is necessary. For example, because the IL-2 Interleukin-2 use of MSCs for treating HD has been predominantly IL-4 Interleukine-4 used in preclinical trials, researchers should be cogni- iPSCs Induced pluripotent stem cells zant that the subpopulation of cells being used is the LRRK2 Leucine-rich repeat kinase 2 gene direct function of the number of passages these cells MBD Methyl binding domain MBS Myelin basic protein have undergone. Though higher passaged MSCs have MeCP2 Methyl-CpG binding protein 2 gene proven to be highly beneficial in restoring the motor MHC class-II Histocompatibility complex class II symptoms associated with HD, there is also a risk of 5mC 5-Methylcytosine chromosomal abnormalities that would lead to tumor mHtt Mutant huntingtin protein formation or other adverse effects. Therefore, study- miRNA MicroRNA MPTP 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ing the epigenetic mechanism of MSCs to provide an MS Multiple sclerosis effective cell replacement therapy is necessary. Some MSC Mesenchymal stem cells diseases, such as RTT have an epigenetic basis, which NRSF Neuron-restrictive silencer factor makes them excellent candidates for the yet-to-be- NSCs Neural stem cells developed epigenetic-driven stem-cell replacement PAD 2 Peptidyl-arginine deiminase 2 PD Parkinson’s disease therapies. As such, a mutation corrected RTT-iPSC cell PSP Inhibitory post-synaptic potential line for the transplantation may prove to be a prom- REST Repressor element-1 silencing transcription factor ising therapeutic approach. Similarly, identifying the RTT Rett syndrome

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V. Factors Influencing Epigenetic Changes