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Astrocytes lost in translation
Dooves, S.
2020
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Dooves, S. (2020). Astrocytes lost in translation: From novel Vanishing White Matter models to the first therapeutic strategies.
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CHAPTER 1
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The signaling in the brain is performed by nerve cells or “neurons” (Figure 1). Neurons have long protrusions (“axons”) to communicate with other neurons or the rest of the body. These axons form networks throughout the brain, and are insulated by a fatty substance (“myelin”) to increase signal conduction velocity. Besides neurons the brain is populated by glial cells (Figure 1). There are 3 main types of glial cells: astrocytes, oligodendrocytes and microglia. Astrocytes are supportive cells that are essential to brain functioning. They are involved in neuronal synaptic transmission, maturation of other brain cells, the formation of a barrier around blood vessels (blood-brain-barrier) and response to brain damage. Oligodendrocytes form the myelin around the axons. Astrocytes and oligodendrocytes together are also called the “macroglia”. Microglia are blood-derived immune cells that migrate into the brain before birth. The networks that are formed by the axons have a whitish color due to the myelin. For this reason, the brain can be divided in “grey matter” and “white matter”; the grey matter are areas rich in neuronal cell bodies, the white matter are areas rich in axons and myelin. Glial cells are present in all brain areas, but oligodendrocytes are more abundant in white matter, and astrocytes in the white matter are of a different subtype than those in the grey matter. The health of the white matter is important for the functioning of the brain. As myelin is important for the speed of signal conduction, white matter abnormalities often primarily affect motor skills rather than cognitive functioning. However, white matter abnormalities are reported in many diseases ranging from schizophrenia (1, 2) to more classic
white matter disorders as multiple sclerosis (3, 4). Genetic disorders that affect the white
matter (leukodystrophies) often affect children, have a devastating impact on patients’ quality of life and treatments are currently lacking. The aim of this thesis is to advance development of treatment options for a rare but severe brain white matter disorder called “Vanishing White Matter” (VWM).
Blood vessel
Neuron
Myelin
White Matter Astrocyte Grey Matter Astrocyte
Microglia
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Vanishing White Matter
Vanishing White Matter (VWM; OMIM 603896) is a severe leukodystrophy, a genetic disease affecting the white matter areas of the brain. All leukodystrophies are rare, but together they have an incidence of about 1:7500 live births (5). VWM is one of the more
common leukodystrophies; about 2-2.5% of the leukodystrophy patients have VWM (5-7).
VWM patients show progressive neurological deterioration like ataxia and spasticity, while cognition is usually relatively spared (8). There are different variants of VWM recognized
based on age of onset, which is inversely correlated with disease severity. The classic form of VWM has an onset in early childhood and initial signs are often ataxia and to a lesser degree spasticity (9). A typical characteristic of VWM is that patients may have episodes of
rapid deterioration, which can be triggered by stressors as febrile infections, head trauma or acute fright (9-11). These episodes can be associated with a decrease in consciousness
and can lead to coma and/or death. After the episodes recovery may occur, but is usually incomplete (9). Besides the episodic fast deterioration, VWM is generally slowly progressive.
Magnetic Resonance Imaging (MRI) is diagnostic in the classic form of VWM: patients show diffuse abnormal signal throughout the cerebral white matter with in the corpus callosum a typical sparing of the outer rim, while the inner rim is affected (12). Over time the cerebral
white matter shows rarefaction to cystic degeneration while the grey matter is spared (8). The
cerebellum and spinal cord can be affected, but to a lesser extent (13).
Severe forms of VWM may show onset of symptoms in the perinatal period or in the first year of life. Cree leukoencephalopathy is a VWM variant that was initially recognized in Cree indigenous people (14). It has an onset in the first 3 months of life and is fatal before the
age of 2 years (9). The most severe, pre- or perinatal onset forms often show involvement of
multiple organs, including involvement of kidneys, liver, spleen, pancreas, eyes and ovaries
(15). MRI can show swelling of the white matter, and the gyral pattern may be immature (15).
Milder variants of VWM have a juvenile or adult onset. The oldest recorded onset of VWM is at age 66 (16). The adult onset form may be more difficult to diagnose than the classic form
as the symptoms, disease course and MRI can be less typical. Patients may initially present with a variety of symptoms like seizures (9), migraine (17), psychiatric symptoms (18) or presenile
dementia (19). Episodes with rapid deterioration can occur, but are less common, and the
disease course is much longer than in children (18). Females often present with ovarian failure
or amenorrhea; the ovaries are the second most commonly affected organ in VWM (8). MRI
shows typical white matter involvement, but cystic degeneration is limited. Cerebral and cerebellar atrophy is often observed (18).
VWM is a severe disease affecting the brain white matter with a variable age of onset, which is inversely correlated to disease severity. The classic childhood onset form usually presents with ataxia, but early or late onset patients can present with distinct symptoms and MRI characteristics. Curative treatment options for VWM are lacking and urgently needed considering the severity of the disease.
Figure 1. Types of brain cells. Different cell types can be found in the brain. The neurons are responsible
for signal conduction, which is facilitated by myelin, a fatty substance wrapped around the neuronal processes by oligodendrocytes. Astrocytes are important housekeeping cells that contact all other brain cells and the blood vessels. Different subtypes of astrocytes exist. The microglia are the immune cells of the brain.
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Figure 2. The role of eIF2B in protein synthesis. For initiation of translation, eIF2-GTP, met-tRNAi and
the 40S ribosome bind the mRNA. Upon recognition of the start codon the 60S ribosome binds, GTP is hydrolyzed, eIF2-GDP is released from the complex and protein synthesis is started. For each new round of translation, the GDP bound on eIF2 needs to be exchanged for a GTP. This reaction is catalyzed by eIF2B.
Genetic background of VWM and the effect on protein translation
The first step in understanding the disease mechanisms of a genetic disorder is to look at the disease causing mutations. VWM is caused by recessive mutations in the genes encoding any of the subunits of eukaryotic translation initiation factor 2B (eIF2B) (20, 21). There are 5 subunits,
eIF2Bα-ε, encoded by the EIF2B1-5 genes. EIF2B is involved in translation initiation; the start
of protein synthesis (see Figure 2). Proteins are synthesized according to a messenger RNA (mRNA) template by ribosomes that link amino acids supplied by the transfer RNAs (tRNA). Translation is usually started from the AUG start codon and recognition of the start codon is aided by the ternary complex and various initiation factors (22). The ternary complex is
formed by eIF2-GTP and a methionine-charged tRNA initiator (met-tRNAi), and binds the small ribosomal subunit (23). Upon recognition of the start codon, the large ribosomal subunit
binds, the GTP is hydrolyzed to GDP, eIF2-GDP is released and protein synthesis is started
(24). In order to start another round of translation eIF2-GDP needs to be reactivated. This is
where eIF2B comes into play. The eIF2-GDP is exchanged for eIF2-GTP by eIF2B, which is a guanine nucleotide exchange factor (GEF) (23). The GEF activity of eIF2B is essential for protein
synthesis in each cell and as such for the vitality of each cell. Under certain circumstances, the eIF2B GEF activity becomes a rate-limiting factor on protein synthesis. The activity of eIF2B can be regulated by phosphorylation. Several phosphorylation sites that regulate GEF activity on eIF2B are known. One site is phosphorylated under low levels of amino acids by an unknown kinase, which inhibits eIF2B activity (25). A second site is phosphorylated by
glycogen synthase kinase 3 (GSK3) when insulin levels are low and is inhibitory for eIF2B activity as well (25). The rate-limiting role of eIF2B on protein synthesis is most studied under
VWM mutations eIF2B eIF2 eIF2 eIF2 eIF2 GTP GTP GDP GDP 60S ribosome
met-tRNAi start codon met-tRNAi
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conditions of cellular stress. There are kinases that phosphorylate eIF2 on the α subunit after amino acid starvation, viral infection or endoplasmic reticulum (ER) stress (Figure 3) (24).
Phosphorylated eIF2α (p-eIF2α) inhibits eIF2B’s GEF activity leading to a decreased amount of eIF2-GTP and thereby inhibiting general protein synthesis (26). Downregulation of protein
synthesis can promote cell survival by limiting accumulation of denatured or toxic proteins and preserving cellular energy.
The ER is involved in the synthesis of transmembrane and secretory proteins, lipid production and calcium storage (27). Unfolded or misfolded proteins can have detrimental
effects on ER functioning through, for example, the formation of aggregates, and protein folding is highly controlled (28). When the amount of un- or misfolded proteins is too high,
the unfolded protein response (UPR) is activated. This response is a protective mechanism that lowers global protein synthesis while increasing the amount of chaperone proteins and folding enzymes in the ER (27). There are three UPR branches: the ATF6 (activating
transcription factor 6) and IRE1 (inositol-requiring enzyme 1) branch increase mRNA degradation, ER folding capacity and protein degradation (26). The third branch involves
dimerization of PKR-like ER kinase (PERK), which then phosphorylates eIF2 on the α subunit. Translation of specific mRNAs containing upstream open reading frames or internal ribosomal entry sites is upregulated when the amount of eIF2-GTP available is low (23).
An example is activating transcription factor 4 (ATF4), which activates a gene expression program that helps overcoming ER stress and provides a negative feedback loop leading to eIF2 dephosphorylation by protein phosphatase I (PP1), regulated through CHOP (C/EBP homologous protein) and GADD34 (growth arrest and DNA damage inducible 34) (Figure 3) (18) (23). Cellular stressors as hypoxia or amino acid starvation can induce phosphorylation of
eIF2 on the α subunit as well; the signaling pathway dependent on eIF2 phosphorylation is termed the integrated stress response (ISR).
Many different eIF2B mutations are known, mostly in the EIF2B5 gene, which
encodes the catalytic eIF2Bε subunit. The majority of patients are compound heterozygous, meaning that they inherited two different mutations in the same subunit gene. There is a genotype-phenotype correlation, with certain mutations consistently causing a severe disease while others are generally mild (8, 29). There is a lot of variation between patients, even
between siblings (30), suggesting a role for the environment or genetic disease modulating
factors. Most VWM mutations decrease the GEF activity in an eIF2B activity assay, but the level of residual GEF activity is not correlated with disease severity (31, 32). Some mutations
that are associated with severe disease do not affect GEF activity at all (32), and mutations
that increase the eIF2B GEF activity have also been reported (33). These results might be
biased by the current available eIF2B GEF activity assays, that measure only GDP release and might overlook other essential steps for eIF2B GEF activity (34). Better eIF2B GEF activity
assays are currently under development. Additionally, it is possible that eIF2B might have different, currently unknown functions that are affected by these mutations.
The decreased eIF2B activity that is found in most patients does not correlate with the reported normal levels of protein synthesis in patients’ lymphoblasts (35) or fibroblasts (36), but human cells carrying VWM mutations show a stronger response to ER stressors (35-38). Most of these studies are either performed in patients’ cells from unaffected tissue
(fibroblasts or lymphoblasts), or in cell lines that are transduced to express mutated eIF2B. The GEF activity of eIF2B or global protein synthesis rates in brain cells derived from patients has not been studied. Activation of all branches of the UPR is observed in post mortem brain tissue of VWM patients (40).
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Figure 3. Mechanisms and feedback loop on eIF2 phosphorylation. A number of stressors can induce
kinases that phosphorylate eIF2. The activity of eIF2B is inhibited by p-eIF2α, leading to a lower availability of eIF2-GTP and a decrease in general protein synthesis. Specific mRNAs are preferentially translated when the level of eIF2-GTP is low. One of these mRNAs is the transcription factor ATF4, that activates a gene program to overcome situations of cellular stress. One of the downstream proteins of ATF4 is CHOP, which in turn promotes transcription of GADD34. GADD34 binds protein phosphatase 1 (PP1) and can dephosphorylate eIF2. Additionally, the constitutive repressor of eIF2 phosphorylation (CReP) can bind PP1 and dephosphorylate eIF2. The activity of eIF2B can also be regulated by phosphorylation of eIF2B itself, which is a downstream effect of for example low insulin levels through GSK3.
To conclude, VWM is caused by mutations in eIF2B, a protein complex involved in protein translation and the cellular stress response. Most (but perhaps not all) VWM mutations affect the GEF functioning of eIF2B, although the protein synthesis rate is typically unaffected. How eIF2B mutations affect GEF activity and protein synthesis in human brain cells is not known and proper model systems to investigate this are lacking.
Cells of the brain white matter are affected by VWM mutations
Although eIF2B functioning is essential for every cell in the body, VWM mutations mainly affect the astrocytes and oligodendrocytes of the brain white matter. Studies in
post-PKR VWM mutations eIF2B eIF2 GTP eIF2 p-eIF2α ATF4 ATF4 PERK PERK PERK PERK BiP
Decrease in general protein synthesis when eIF2-GTP is low
CHOP
GADD34
ER stress Viral infection Amino acid starvation
GCN2 PP1 PP1 CReP GSK3 Low insulin Chaperone proteins
Amino acid synthesis Antioxidant response
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mortem brain tissue show that the white matter is cavitated, consistent with what can be observed on MRI (9). In more preserved white matter areas, oligodendrocyte progenitor cells
(OPC) are increased in number, but do not mature into myelinating oligodendrocytes (41, 42). A number of oligodendrocytes have a “foamy” appearance with vacuoles inside their
cytoplasm (9). In areas that are still myelinated, the myelin often shows vacuolization. Axons
are relatively spared but are completely lost in cavitated areas. Astrocytes have an abnormal morphology, with short and blunt processes, and express markers related to immature astrocytes, like CD44, Nestin and the δ isoform of glial fibrillary acidic protein (GFAP) (41).
Both oligodendrocytes and astrocytes show upregulation of proteins involved in the cellular stress response (40). The normal response of astrocytes to tissue damage is reactive gliosis,
which is an initially beneficial processes that functions to limit tissue damage and offers neuroprotection (43). Reactive astrocytes show progressive hypertrophy, proliferation,
process extension and alterations of gene expression; all reactive astrocytes upregulate GFAP (including the δ isoform) (43). Astrogliosis in VWM is remarkably meager (9). Microglia
in VWM patients show no abnormalities in morphology or marker expression, but lack a reactive response to the tissue damage much like the astrocytes (9). There is an increase
of high molecular weight hyaluronan (HMW-HA) in the brains of VWM patients compared to controls (44). Hyaluronan (HA) is in the brain mainly produced by astrocytes and it is
known that HMW-HA can inhibit OPC maturation (45). Furthermore, (HMW) HA can inhibit
astrogliosis (46, 47). As both a low number of mature oligodendrocyte and an abnormally low
amount of astrogliosis are observed in VWM, HA is an interesting therapeutic target. Studies on post-mortem brain tissue on patients are very informative, and gave the first clues into the pathomechanisms of VWM. Why the brain white matter is specifically affected by eIF2B mutations needs further study, although not explored within the current thesis.
To further elucidate the disease development and to test possible therapies, it is essential to have proper disease models. Two mouse models for VWM have been developed. The first model developed by Geva et al. (39) contains a R132H point mutation in Eif2b5 corresponding
to the human R136H mutation in EIF2B5 that is known to cause a mild, adult onset form
of VWM. The mice show a subtle phenotype, with no obvious motor defects or limited survival. Some abnormalities are observed, like a decreased GEF activity, abnormal levels of myelin proteins and an increased number of small caliber axons (39). Protein synthesis rates
in the brain are normal (39). Developmental gene expression is delayed (48), and in young mice
there is an increased number of oligodendrocytes and decreased number of astrocytes, which is normalized in 4-month-old mice (39). After treatment with lipopolysaccharides
(LPS), which triggers an inflammatory response, astrogliosis is reduced in Eif2b5R132H/R132H mice (49). Both astrocytes and microglia fail to induce interleukin-6 (IL-6) and interleukin-1β
(IL-1β) after LPS stimulation (49). Furthermore, the Eif2b5R132H/R132H mice do not recover from demyelination induced with a cuprizone diet (50). A proposed hypothesis is that the glial cells
of the Eif2b5R132H/R132H mice are able to cope with normal translational demand, but the cells are unable to cope with the acute demand of increased translation after demyelination or other stressors (49, 50).
Another mouse model for VWM has been proposed by Lin et al. (51). In this mouse
model, the eIF2 kinase PERK can be activated in oligodendrocytes specifically by injection of a synthetic compound called AP20187. The authors show that this activation of PERK leads to an increased p-eIF2α and a decreased protein synthesis, which corresponds to a lower eIF2B activity (51). When the mice are injected with the AP20187 from postnatal day 10 (P10)
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on they show a very strong tremoring phenotype and die at P24 (51). Interestingly, they have
oligodendrocytes that show a similar “foamy” appearance to oligodendrocytes observed in VWM patients (51). Whether this mouse model is representative of VWM can be debated. As
discussed in 1.1.2, not all VWM mutations lower eIF2B activity, and protein synthesis is not decreased in non-brain cells from VWM patients or brains of Eif2b5R132H/R132H mice.
To summarize, the studies on post mortem brain tissue and in the first VWM mouse models show that astrocytes and oligodendrocytes are mainly affected by VWM mutations. Oligodendrocytes show increased proliferation with a decreased maturation. Astrogliosis is affected, and mice are unable to recover from severe demyelination. At least part of the VWM pathology might be mediated by increased levels of HA. To further understand the pathomechanisms of VWM and test possible treatments we are in need of a good mouse model that replicates all aspects of the human disease VWM.
Stem cell therapy
Cell replacement therapy is a promising option for leukodystrophies. To generate cell populations for graft studies, different origins and types of stem cells have been explored. Stem cells have two important characteristics: stem cells can (1) self-renew and (2) differentiate into another (more mature) cell type (potency). Stem cells can be classified according to their potency. A totipotent stem cell is a cell that can differentiate into all cells of the body and the extra-embryonic tissue; these are fertilized eggs and cells from the first few divisions. A pluripotent stem cell is a cell that can differentiate into all cells of an adult body, but cannot make extra-embryonic tissue. Pluripotent stem cells can be derived from the inner cell mass of an blastocyst. In the lab, the pluripotent stem cells derived from embryonic tissue, called embryonic stem cells (ESCs) can be cultured and maintained in a pluripotent state with the addition of a number of defined factors (52). Removal of these
factors induces spontaneous differentiation into all three germ layers. Many protocols have been developed to differentiate ESCs to specific cell types or lineages, to study developmental processes or as a possible cell source for cell replacement therapy. However, as ESCs are derived from embryonic tissue, ethical concerns are associated with their use. Undifferentiated pluripotent stem cells form tumors, so pluripotent stem cells will need to be differentiated to a more restricted cell type before transplantation.
A multipotent stem cell can differentiate into multiple cell types but with a lineage restriction. Multipotent stem cells are abundant in the body; all organs retain multipotent stem cells throughout life to replenish tissue cells. In the brain there are multipotent neural progenitor cells (NPCs) that form during embryonic development and generate neurons and glial cells. In adults NPCs persist in the subventricular zone next to the lateral ventricle and in the subgranular zone in the hippocampus (53). These adult NPCs are slowly dividing cells that
continue to form new neurons and glial cell throughout life (53). Stimulation of endogenous
NPCs in disease or injury is a possible therapeutic approach, although depletion of the NPC pool is implicated as a main contributor to brain decline during ageing (53). For this reason
NPCs might be more useful as an in vitro cell source that can be expanded and differentiated
before transplantation. NPCs can be derived from fetal tissue or by differentiation of pluripotent stem cells, which is discussed in more detail below. Multiple kinds of stem cells exist, which differ in their potency. Stem cell therapy for VWM would likely involve a neural or glial progenitor cell and not undifferentiated pluripotent stem cells.
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Induced pluripotent stem cells
In 2006 and 2007 it was reported by the lab of Yamanaka that they were successful in generating pluripotent stem cells from fibroblasts, a mature cell type abundantly present in the skin (54, 55). Previously, it was thought that terminally differentiated cells could not
be brought back to an undifferentiated state. The initial protocol to derive these induced pluripotent stem cells (iPSCs) from fibroblasts was fairly simple, using viral overexpression of just 4 genes (OCT4, KLF4, C-MYC, SOX2) to induce pluripotency. By now multiple additional
methods have been developed, such as non-integrative gene delivery methods, episomal plasmids or small molecules (56). Multiple somatic cell types can be reprogrammed; fibroblasts
are often used and easy to culture, but require a skin biopsy. Other possible sources for cells are blood, urine or hair, which are more convenient for patients (57). The iPSCs opened up a
whole new dimension to stem cell therapy. It is now possible to take a skin biopsy from a patient, make iPSCs and derive the necessary cell type from patient specific stem cells. This way the chances of rejection after transplantation and the ethical problems associated with ESCs are reduced.
Next to iPSC applications for stem cell therapy, the invention of iPSCs opened new ways to develop in vitro disease models and to test new drugs. By generating iPSCs
from patients with disease causing mutations and differentiating the iPSCs into disease relevant cell types, human cellular dysfunctions can be studied in a cell culture dish. Cellular phenotypes could give insight into disease mechanisms. Using patient-derived iPSCs, researchers have shown abnormalities in astrocytes from amyotrophic lateral sclerosis (ALS), Alzheimer disease (AD) and Huntington disease (HD) (58). Another example is a study
in which an increase in ER stress in oligodendrocytes of hypomyelinating disorder Pelizaeus-Merzbacher disease (PMD) was found (59). Disease modeling with iPSCs can lead to new drug
targets, which can first be tested on patient-derived brain cells. This will increase the chances of finding treatments that are effective in humans and lowers the amount of experimental animals needed for preclinical testing.
iPSC tools have advanced the stem cell applications enormous and are now widely explored for applications in regenerative medicine, disease modeling and drug development. Current challenges of the iPSC field lie in developing standardized efficient differentiation protocols to the cell types of interest with low variability, including generation of functional neural-specific cell types.
Generation of patient-derived glial cells
Many differentiation protocols to generate glial cells from pluripotent stem cells have been developed for both mouse and human pluripotent stem cells (58, 60-64). Most protocols
mimic normal development, based on studies in animal model systems. The developmental stages can be divided into 4: (1) neural induction; (2) neural patterning; (3) gliogenic switch; and (4) terminal differentiation (58). After the neural induction and neural patterning
phase, cultures contain NPCs that are patterned to an approximate brain region. Initially NPCs mainly produce neurons, which is consistent with normal development in which neurogenesis precedes gliogenesis. Over time and with fibroblast growth factor 2 (FGF2) stimulation, NPCs will make the gliogenic switch and start producing glial cells. Terminal differentiation of a mature astrocyte can be promoted by culturing in the presence of ciliary neurotrophic factor (CNTF), bone morphogenetic proteins (BMPs) or serum (58).
Oligodendrocyte differentiation protocols usually include patterning with sonic hedgehog (SHH) and retinoic acid (RA) inducing a ventral posterior spatial identity (64). During
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development, the first wave of oligodendrogenesis occurs ventrally and is SHH dependent, so most differentiation protocols mimic this first wave of OPC generation (65). Terminal
differentiation of oligodendrocytes is usually promoted with insulin growth factor (IGF1) and triiodothyronine (T3) (64). Oligodendrocyte differentiation protocols generally lead to a
mixed culture with astrocytes present, unless sorting or immunopanning steps are used to isolate oligodendrocyte lineage cells (64).
Both the protocols for the generation of astrocytes and oligodendrocytes from iPSCs are time consuming and might take between 4 and 9 months to generate mature glial cells from human iPSCs. Different studies have looked at ways to generate glial cells in a shorter time frame, for example by culturing under low oxygen or reducing the time it takes to induce neural stem cells from iPSCs (64). Another possible source of glial cells is by
transdifferentiation. With transdifferentiation somatic cells are not first reprogrammed to iPSCs but directly converted into a precursor population or in the cell type of interest (66).
Advantages of transdifferentiation over iPSCs include shorter differentiation protocols and possible smaller chances of tumorigenesis when used for therapeutic purposes. However, transdifferentiated cells might retain more epigenetic memory. More research is needed to identify the optimal factors to generate glial cells from fibroblasts or other somatic cell sources (66).
Overall, multiple protocols exist to make glial or glial progenitor cells from patient’s own cells, either through a pluripotent state or by direct transdifferentiation of somatic cells. These cell populations are of interest for therapeutic purposes, as they lack ethical issues associated with ESCs and have a low chance of rejection after transplantation.
Stem cell-based therapy
Cell replacement therapy is seen as a promising treatment option for a range of different disorders. For leukodystrophies cell replacement therapy is focused on transplantation of healthy macroglial cells, that can replace the aberrant host macroglial cells. These glial cells can be derived from iPSCs, but also from other (stem cell) sources, like fetal tissue or ESCs
(67). If patient-derived iPSCs are used, the genetic defect will have to be corrected, but this is
feasible with recent advantages in gene correction methods like CRISPR-Cas (52, 68).
Glial cell replacement therapy has been studied for hypomyelinating disorders, like PMD. PMD is a leukodystrophy caused by mutations, most often duplications, of the proteolipid protein 1 (PLP1) gene, encoding one of the major myelin proteins (69).
Interestingly, null mutations cause less severe disease than duplications and missense mutations, indicating that a lack of PLP1 protein is less severe than overexpression or mutated PLP1 protein (69). Hypomyelinating diseases are often modeled by natural
occurring rodent mutations; an example is the shiverer mice which lacks myelin basic protein (MBP) and as a consequence lacks functional myelin (70). Transplantation of
human glial progenitor cells can rescue myelin defects and increase survival in shiverer mice (71, 72). Furthermore, transplantation of human glial progenitor cells improves
the phenotype in a more specific Plp1 overexpressing mouse model for PMD (73).
These successful preclinical studies led to the first Phase I/II human clinical trial using cell replacement therapy for PMD. In this study fetal tissue derived cells were transplanted in the brains of four PMD patients (74). The one year follow-up showed safety of
the procedure, but efficacy could not convincingly be established (74). For the continuation
of research into glial cell replacement therapy it is very promising that no serious adverse effects were observed. Future studies with larger patient groups are necessary to establish
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Figure 4. Possible uses for stem cells in VWM. Somatic cells can be isolated from patients and
reprogrammed into iPSCs. These iPSCs can be differentiated into glial cells that can be used for disease modeling and drug screening. Alternatively, iPSCs can be genetically corrected and differentiated into glial progenitor cells that can be used for transplantation.
efficacy of cell therapy for PMD and other leukodystrophies.
Noteworthy, although (iPSC-based) neural cell replacement is being explored, stem cell therapy in the form of hematopoietic stem cell transplantation (HSCT) has been performed for many years. Since the 1950s (75) cancer patients that underwent radiation
therapy received HSCT. The number of eligible diseases has grown. HSCT is effective for a group of leukodystrophies that belong to the lysosomal storage diseases: these patients have mutations in lysosomal enzymes leading to accumulation of enzyme substrates (67).
HSCs can differentiate into monocytes and microglia and infiltrate the brain tissue, were they can provide the missing enzyme to relieve the load of accumulated proteins and have immunomodulatory effects (76). HSCTs is used to treat metachromatic leukodystrophy (MLD),
and globoid cell leukodystrophy (GLD), but is generally only successful when performed early in the disease course, sometimes even before onset of symptoms (76). In early disease stages,
HSCT is also effective for the peroxisomal disorder adrenoleukodystrophy (ALD) through unknown mechanisms, probably also by supplementing healthy microglia.
In conclusion, different studies have shown promise for stem cell therapy for leukodystrophies. We hypothesize that glial cell replacement therapy is a promising option for VWM patients (Figure 4).
Isolate somatic cells from patient
Reprogram somatic cell to iPSC
Use gene targeting to correct VWM mutation Differentiate into glial cells
for disease modeling and drug screening Test drugs
in patients
Differentiate into glial progenitor cell
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Outline
VWM is a severe disease and patients are in need of treatment options. It is known that VWM is caused by eIF2B mutations and that mainly the astrocytes and oligodendrocytes of the brain are affected. The aim of this thesis is to explore and advance new treatment options for VWM. Additionally, this thesis aims at developing new model systems for VWM using a new mouse model and iPSC technology. Good models are essential to test treatments and to study disease causing mechanisms. How eIF2B mutations cause VWM is beyond the scope of this thesis, but future studies can use the models developed in this thesis for more in depth studies of VWM pathomechanisms.
To test therapies we need representative animal models. Two mouse models for VWM have been developed previously, but these models do not replicate all aspects of VWM in patients. Chapter 2 describes new mouse models for VWM. We used mutations
that are known to cause a severe form of VWM in patients. Mice with mutations in Eif2b4
or Eif2b5 showed many similarities to patients, like gait abnormalities, shortened lifespan
and affected astrocytes and oligodendrocytes. By crossing these mice into Eif2b4/Eif2b5
double mutant mice we obtained mice with a very severe phenotype that can be used as a model for early onset VWM. We used these mice to study the development of VWM pathology over time, and used in vitro studies to elucidate the influence of astrocytes on
oligodendrocyte pathology.
To generate glial cell populations which can replace the affected cells in VWM, we explored mouse and human iPSC tools to generate glial cell types (Chapter 3). Mouse
iPSCs and patient and control human iPSCs were differentiated into astrocytes and oligodendrocytes. Protocols were developed to culture white like and grey matter-like astrocytes from iPSCs. We could confirm the central role for astrocytes that we observed in chapter 2 with mouse and human iPSCs. Additionally, we identified pathways that were specifically affected in human VWM white matter-like astrocytes. In the future these iPSCs can be used for drug screening, studies into disease mechanisms and transplantation studies.
To evaluate treatment options, e.g. recovery after cell grafting, we are in need of good disease markers in our VWM mouse models. In Chapter 4 we present new disease
markers for VWM to validate treatment efficacy. Mice were treated with Guanabenz, a compound effecting the cellular stress response. Based on a number of quantitative disease markers the treated animals showed improvements after Guanabenz treatment
To generate proof-of-concept for stem cell therapy, we transplanted VWM mice with primary mouse glial progenitor cells. In Chapter 5, we explored the regenerative
capacity of three different populations of glial progenitor cells. After injection we compared
the different populations on their survival, integration, maturation and ability to improve the VWM phenotype. A number of VWM mice improved after cell injection. This is the first study to show positive prospects of cell therapy for VWM.
Earlier studies showed that HA levels are increased in the VWM brain. As this could potentially affect survival of glial cell grafts, we explored HA modulating treatments in
Chapter 6. Although lowering hyaluronan levels on itself did not improve VWM pathology,
no adverse effects were observed. By lowering hyaluronan levels in the brain, the survival, migration and differentiation of transplanted cells could be improved. Future studies can combine hyaluronan lowering compounds with cell therapy.
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of stem cell therapy. In Chapter 8 I discuss the implications of the results of this thesis for
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