technology to neuromuscular disorders
Sterrenburg, P.J.E.
Citation
Sterrenburg, P. J. E. (2007, January 18). Application of microarray-based gene expression technology to neuromuscular disorders. Retrieved from https://hdl.handle.net/1887/8914
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Chapter 6
Discussion
6.1 Gene expression profi ling
6.2 In vitro myoblast diff erentiation as a model for studying human muscle regeneration
6.3 Impaired regeneration in DMD muscle 6.3.1 Intrinsic versus extrinsic factors 6.3.2 Implications for therapy
6.4 Contribution of intranuclear inclusions to the OPMD phenotype
6.4.1 Cellular model for OPMD and cellular eff ects of the presence of inclusions 6.4.2 Composition of inclusions and cellular eff ects
6.5 Muscle-specifi c formation of intranuclear inclusions in OPMD 6.5.1 Muscle specifi c protein or RNA
6.5.2 Profi brillar structures that cannot be cleaned up by the proteasome 6.5.3 PABPN1-concentration dependent initiation of inclusion formation 6.5.4 Combination of the theories
6.6 Conclusion
6.1 Gene expression profi ling
The fi rst studies described in this thesis (Chapter 2-4) were performed in ‘the early microarray days’ using home-made cDNA microarrays representing 5000 genes. Back then, cDNA arrays often contained smaller sets of genes because it was diffi cult to achieve high-quality, error-free preparation and printing of large series of clones. For the experiments described, we designed a so-called ‘muscle-related’ set of genes and ESTs previously reported to be involved/expressed in ‘muscle’ in the broadest sense of the word. The use of such a subset puts a bias in the results of the gene expression profi ling experiments and unexpected changes, i.e. of genes not normally expressed in muscle, will probably go unnoticed. Furthermore, after data analysis, the top list of differentially expressed genes needs to be sequenced to confi rm the identity of the printed clones, since information on the clones is sometimes not available or not correct due to contamination in the plate. In spite of the limitations of the array, interesting results were obtained and after sequencing, only one in 25 clones appeared to have a different identity than originally reported.
With technology maturing, synthetic 50-60 mer oligonucleotides became cheaper and larger gene sets often representing the entire genome, became available. In Chapters 3 and 5 these arrays were used, immediately proving the usefulness of a whole genome array; the new pathway described to be involved in OPMD, would probably not have been detected on a dedicated array because the genes in this functional class were not suffi ciently represented.
Gene expression profi ling using microarrays has great advantages. Due to its unbiased nature and comprehensiveness, the technique can generate new hypotheses and provide new insights. However, the technique should be applied with care. The resulting amount of data can overwhelm users and the analysis and interpretation is diffi cult. Important hints are easily missed in these experiments. With improved annotation and new data analysis programs and algorithms continuously being developed, it is possible that an experiment performed in 2000, can still reveal new insights in 2006. With biosemantics emerging and new developments rapidly occurring in this fi eld, interpretation of microarray data will become much easier in the future. Intelligently summarizing literature and other web-published, human-annotated information on a large scale, by text mining and meta-analysis, will provide us with links, relationships, associations and patterns that were not recognised before (discovery of implicit knowledge).
6.2 In vitro myoblast differentiation as a model for studying human muscle regeneration
Multiple studies have characterized gene expression during myoblast differentiation on a genome-wide scale (1-4). Different cell culture model systems with different time spans have been used for this. Two studies used C2C12 mouse myoblast cells; Delgado et al. investigated the fi rst 24 hours of differentiation and Tomczak et al. investigated 10 days of differentiation.
Both found several clusters of genes up- or downregulated during differentiation. Another approach was followed by Bergstrom et al. who used mouse fi broblasts containing a fusion protein of MyoD and an estrogen receptor binding domain and induced myogenesis by addition of β-estradiol to the cells (1,5). They particularly looked at promoter-specifi c regulation of MyoD binding and found 82 genes to be directly regulated by MyoD. In Chapter 3 a gene expression study using primary human myoblast cultures is described. Analysis of primary human cell cultures is probably the most representative way to study human muscle cell
103
differentiation. Not only because it is a human model system, but also because the cell cultures are ‘contaminated’ with other proliferating cells present in the muscle. This mixed population of mainly myoblasts and fi broblasts better represents the original environment of muscle and in this way, also signaling interactions between the different cells are included, as they may take place in the muscle itself. A disadvantage of this system is the heterogeneity (myogenicity %) of the different cultures. This makes it necessary to test more cultures to fi nd the common denominator. Nevertheless, in our study 145 genes were found to be differentially regulated upon induction of muscle cell differentiation, including 86 previously undetected genes. Possibly these are specifi c for human myogenesis or refer to the above mentioned signaling interactions. The use of siRNA or overexpression experiments can shed more light on their specifi c function in muscle cell differentiation.
This model system can also be used to study muscle regeneration. An important alternative model is induction of degeneration in myofi bers by injection of a toxic agent (eg. cardiotoxin) in mouse/rat muscle (6). Differential gene expression is then determined in the whole muscle.
The disadvantage of this model, when studying the actual regeneration by myoblasts, is that in a whole muscle biopsy the contribution of myoblast gene expression is too small compared to other cell types and can not specifi cally be detected. In this perspective, the use of myoblast cultures and fusing them in vitro is a good model system to study the early gene expression changes of the key players in muscle regeneration. Using this culture system it became clear that myoblasts fi rst differentiate into fusion-competent myoblasts before the actual fusion of the cells into myotubes (Chapter 3). Almost no changes in expression profi le are visible after day 4, while the actual fusion of the cells occurs mainly after day 6. At the same time, this points to an important limitation of gene expression profi ling; translational and post- translational regulatory mechanisms will be missed and it can be diffi cult to link transcription profi les with visible changes. Protein studies will be of additional value in such cases.
Still, the expression profi les of in vitro differentiating myoblasts are very informative and can be used in combination with gene expression profi les from myogenic cell cultures of muscular dystrophies to fi nd targets that can stimulate regeneration. Especially in Duchenne muscular dystrophy this approach of using human myoblast cultures is useful, because regeneration in DMD patients differs from that of the mouse model for DMD, the mdx mouse (7). DMD muscle fails to actively regenerate, while mdx muscle shows an almost complete regeneration and a less severe phenotype in comparison with DMD patients (8,9). As a consequence, methods or model systems like the mdx mouse model or mouse myoblast cell cultures can not be used to study the defective regeneration in DMD patients. Furthermore, the cells used in the in vitro study are activated satellite cells that normally reside in the adult human DMD muscle and are activated there upon muscle injury. Activation in culture and stimulating them to differentiate and fuse thus mimics the real situation closely. The next paragraph points out differences in expression between healthy and DMD primary myoblast cultures which have not previously been recognized using other model systems.
6.3 Impaired regeneration in DMD muscle
One of the outstanding issues in Duchenne muscular dystrophy is whether the defective regeneration is caused by replicative senescence due to a too rapid turnover, by a defect in differentiation, or by extrinsic processes like continued (e.g. immune-mediated) muscle degeneration. Many studies have been performed to address this issue, but results are neither consistent nor conclusive (10-13).
In Chapter 4 this controversy about DMD muscle cell regeneration has been addressed experimentally in a gene expression profi ling study using human primary myoblast cultures of DMD patients. In myoblasts the full-length DMD gene is not yet expressed and in the DMD cell cultures used, production of the embryonic Dp71 isoform (14) is not different from healthy muscle cell cultures because the mutation is upstream of the promoter. Taken this knowledge into account, no differences in gene expression are expected between healthy and DMD myoblasts. However, morphological differences between healthy and DMD myoblasts were previously observed by electron microscopy, suggesting that the satellite cells have received signals from the defective muscle (15).
Figure 1. Schematic representation of the Notch signaling pathway (left) and BMP signaling pathway (right). The BMP signaling pathway can interact with the Notch pathway to stiumulate myogenic proliferation (dotted line).
6.3.1 Intrinsic versus extrinsic factors
Gene expression differences between healthy and DMD myoblasts, in particular those described in Chapter 4, support the morphological differences found. The observed downregulation of FGF2 in DMD cell cultures appears to be a downregulation in fi broblast FGF2 expression pointing to the signaling interaction between myoblasts and their surrounding cells. Previously it was shown that there are anti-proliferative factors (eg. TGF-β1) produced by DMD fi broblasts (16). The lower expression of FGF2 we observed in DMD cell cultures could also have a negative effect on the proliferation capacity of the myoblasts because this factor is known to stimulate replication (17). Future experiments which specifi cally focus on FGF signalling between fi broblasts and myoblasts can study in more detail how this
105
Notch1 ICD
BMP4
proteolytic processing events (presenilins)
Notch1 ICD
translocation to nucleus CSLinteraction
Regulate transcription of target genes Hes and Hey
(related bhlh transcription regulators)
Blocks differentiation of myogenic cells Stimulates proliferation of myogenic cells
Type II receptor
Phosporylation of Type I receptor
Phosphorylation of SMAD1
SMAD1 + SMAD4
Regulation of specific targets Ligand (Delta)
Notch1 transmembrane
protein
interaction
Notch signaling pathway BMP signaling pathway
interaction is exactly regulated.
This study also shows that both AQP1 and BMP4 are signifi cantly upregulated in DMD myoblasts. The functional role of AQP1 in this process is unknown, further research is necessary to explore this. The mechanism by which BMP4 probably operates, is through interfering with the Notch signaling pathway. During regeneration, the Notch signaling pathway is regulated very tightly (Figure 1, left side). Notch is fi rst activated by its ligand, Delta. Then the intracellular domain of Notch is cleaved by presenilins and translocated to the nucleus. Subsequently, by interaction with CSL, it activates a family of transcription factors (Hes, Hesr), which stimulate proliferation of the myoblasts and delay myogenic differentiation (18). After extensive proliferation the Notch signaling is inhibited by Numb, and myoblasts differentiate and become fusion competent (19). BMP4 can also signal via this pathway through its downstream effector SMAD1 which interacts with the intracellular portion of Notch and in this way can also inhibit differentiation (Figure 1, interaction in red) (18).
The upregulation of BMP4 in the DMD cell cultures indicates an increased activation status of the Notch signaling pathway which is supported by an upregulation of HEY1 and HEY2 in the DMD cell cultures. Due to constant regeneration of DMD muscle, this alternative BMP4-Notch pathway is probably triggered to aid in the activation of satellite cells, but at the same time this could inhibit specialization of the myoblasts. As differentiation and fusion of the myoblasts are separate processes, we investigated both indices in BMP4 stimulated cell cultures. It appeared that both differentiation and fusion of DMD myoblasts were signifi cantly reduced, indicating that differentiation is probably inhibited by the BMP signalling and as a consequence, the cells show reduced effi ciency in forming myotubes (Figure 2).
Figure 2. The formation of fusion potent cells after addition of 10 ng/ml BMP4 is more effi cient in normal myoblasts (A) in comparison with DMD myoblasts (B).
Proliferating myoblast
Fusion potent myoblast
Myotube Withdrawal cell
cycle and differentiation
Fusion of myoblasts
Proliferating myoblast
Only few fusion potent
myoblasts
Myotube Addition of
BMP4
Fusion of myoblasts A. Normal differentiation of myoblasts in vitro
B. Hampered differentiation of DMD myoblasts in vitro due to higher BMP4 expression Addition of
BMP4
Withdrawal cell cycle and differentiation
On the basis of the results presented in this thesis, the cause of the defective regeneration in DMD muscle is explained by an imbalance of intrinsic and extrinsic factors. The impaired proliferation by FGF2 downregulation is caused by signaling that originates from fi broblasts (extrinsic) and the impaired differentiation of the DMD myoblasts on the contrary is caused by an elevated BMP4 expression in the myoblasts (intrinsic). Although the latter refers to an intrinsic signaling cascade, the fact that the myoblasts do not yet suffer from a defi ciency in dystrophin, suggests that they are infl uenced by the sick muscle surrounding them (extrinsic).
6.3.2 Implications for therapy
In this case gene expression profi ling not only provides a theory on the cause of the impaired regeneration but also gives information that can be used in treatment of the disease. Future therapy studies can benefi t from this knowledge aiming to stimulate regeneration by interfering in the BMP4 pathway (Chapter 4). For instance, myoblast transplantation will only be successful when these cells also differentiate into mature myofi bers. The administration of a BMP4 inhibitor can possibly further stimulate the cells to differentiate. Future (microarray) experiments will have to show if this effect is positive and what the side-effects are, if any.
In other muscular dystrophies, where research has not (yet) been as extensive as in Duchenne muscular dystrophy, gene expression profi ling can be useful in fi nding new pathways that link the genetic defect to the specifi c phenotype. In Chapter 5 of this thesis, this is shown by an expression profi ling study on Oculopharyngeal muscular dystrophy (OPMD).
6.4 Contribution of intranuclear inclusions to the OPMD phenotype
The most prominent hallmark of OPMD are the intranuclear inclusions (INIs) present in the muscle cells of OPMD patients (20). The frequency of these INIs is correlated with the severity of the disease. Homozygotes have double the amount of inclusions in combination with a more severe phenotype (21). Also, recent studies have shown that reducing the percentage of INIs present in the muscle cells of an OPMD transgenic mouse model (eg. with doxycyclin or trehalose), is associated with delayed onset and attenuation of muscle weakness (22,23).
Studying the formation, composition and cellular effects of these inclusions can therefore contribute to a better understanding of the OPMD phenotype.
6.4.1 Cellular model for OPMD and cellular effects of the presence of inclusions
In Chapter 5 we describe the development of stable myogenic cell lines overexpressing physiologically relevant levels of PABPN1 (wild-type or mutant). The differentiated myotubes have 20-80% intranuclear inclusions and form a good model to study the effects of the presence of these INIs on cellular processes in myogenic cells. Large-scale gene expression analysis revealed a signifi cant downregulation of collagens, collagen synthesis genes and other extracellular matrix (ECM) components. This downregulation is proportional to the percentage of inclusions present in the cells (Figure 3). One of the genes involved in collagen synthesis, PCOLCE was shown to be trapped in the INIs both in the cell model and human OPMD muscle biopsies. As PCOLCE has a possible function in the stabilization of mRNA, it could be that the resulting downregulation of specifi c ECM components is a 107
result of the entrapment of PCOLCE in the INIs. Future experiments such as RNA-IP and co-IP should provide more insight into the nuclear function of PCOLCE and its relation to ECM downregulation. Interestingly, in this respect, a mutation in COL6α resulting in downregulation of Collagen VI causes Bethlem myopathy and Ullrich congenital muscular dystrophy (24,25). Furthermore, Irwin et al. show in a knock-out mouse model of Col6a1 that absence of Col6a1 in the ECM causes a mitochondrial phenotype (26). The downregulation of the whole extracellular matrix in OPMD may therefore also affect this signaling pathway, with a deregulation of mitochondrial processes as a result.
Figure 3. Correlation between inclusion percentage and downregulation of expression of collagens and collagen synthesis genes.
Although the ECM deregulation is clearly visible in the OPMD cell model, there is no downregulation found in the quadriceps of OPMD patients (preliminary data), probably due to the low inclusion percentage (2-5%). However, in most muscular dystrophies an upregulation of the ECM is observed which is absent in OPMD, possibly explaining the initial myopathic appearance of OPMD muscle which only becomes dystrophic in the later stage of disease. (27-30). Moreover, a mitochondrial phenotype has been reported previously in OPMD muscle, with cytochrome-c-oxidase-negative fi bers and aggregates of mitochondria containing paracrystalline inclusions (31-33). The exact signalling pathway between the ECM and mitochondria is not (yet) known, but Rizzuto suggested possible signalling pathways to interact via the integrins or the dystrophin-glycoprotein complex (34). Functional analysis of the microarray data on these specifi c pathways may provide further clues on the link between the ECM and the mitochondria.
6.4.2 Composition of inclusions and cellular effects
Apart from PCOLCE other proteins are also known to be entrapped in the inclusions, like CUGBP1, SFRS3, FKBP1A, HSP70 and HNRPA1 (35-37). The majority of these are involved in RNA splicing, packaging and transport (38-40). In preliminary tests we also detect aberrant splicing in the cellular model for OPMD, as has been reported in myotonic dystrophy (DM) and recently in Facioscapulohumeral muscular dystrophy (FSHD) (41,42).
Inclusion %
0 0.5 1 1.5 2 2.5
Col6a1 P4hb Pcolce Col1a1 Col3a1 Gene
Log2 relativeexpression
0%
23%
32%
77%
Inclusion %
In DM1, an untranslated CTG repeat expansion induces abnormal regulation of RNA- binding proteins which results in alternative splicing of genes (43). These results suggest that disturbance of certain cellular processes, by entrapment of specifi c proteins in the INIs, may contribute to the pathophysiology of OPMD.
Calado et al. (44) show that PABPN1 polyadenylation function is not affected in cell cultures of OPMD myoblasts, but do not mention if these cells contain intranuclear inclusions. The cells are isolated satellite cells originally present in affected muscle and they claim that the satellite cells do not contain INIs. Activation of these cells in culture will not necessarily introduce intranuclear inclusions. So, it cannot be excluded that the polyadenylation function of PABPN1 is hampered in cells containing INIs. They also imply that non-specifi c entrapment of mRNA in the intranuclear inclusions could be a major contribution to cellular dysfunction (44). In our gene expression study however, only 7.5 % of the RNA transcripts show a change in expression level upon inclusion formation which is suggestive for specifi c mRNA trapping in the INIs rather then a general dysfunction in polyadenylation (eg. if the mRNA of one gene is trapped in the inclusions, this can infl uence the transcription of tens to hundreds of other genes). Taking these results into account, it is more probable that entrapment of specifi c proteins and RNAs in the inclusions causes a deregulation of cellular processes by which the cell cannot function in a proper way, rather than non-specifi c entrapment of mRNA in the inclusions, general defects in polyadenylation or just toxicity of the inclusions to the cell (Figure 4).
Figure 4. Schematic representation of possibilities of inclusion formation (top) and how the presence of inclusions can lead to the OPMD phenotype (bottom).
109
Mutated PABPN1
Overexpression of PABPN1
INI formation
Entrapment of specific proteins
or mRNAs
Cellular processes hampered
OPMD PCOLCE
entrapment
ECM components downregulated
Mitochondrial dysfunction
Clean-up of mutated PABPN1 by proteasome
Overexpression of PABPN1 reaches certain threshold
Proteasome cannot keep up with clean-up
ageing Muscle cells
Mutated PAPBN1
High/elevated expression of muscle specific
mRNA
+
Entrapment proteins involved in RNA
processing
Alternative splicing of specific transcripts
Table1: Genes highest expressed in quadriceps skeletal muscle tissue
Name Sequence Description Chr. Loc. GeneID
AMPD1 # Adenosine monophosphate deaminase 1 (isoform M) 1p13 270
APOBEC2 Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 2 6p21 10930
ART3 # ADP-ribosyltransferase 3 4p15.1-p14 419
ATP2A2 ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 12q23-q24.1 488 ATP5E ATP synthase, H+ transporting, mitochondrial F1 complex 20q13.32 514
CASQ1 Calsequestrin 1 (fast-twitch, skeletal muscle) 1q21 844
CKM Creatine kinase, muscle 19q13.2-q13.3 1158
CMYA5 Cardiomyopathy associated 5 5q14.1 202333
COX6A2 Cytochrome c oxidase subunit VIa polypeptide 2 16p 1339
COX7A1 Cytochrome c oxidase subunit VIIa polypeptide 1 (muscle) 19q13.1 1346
FHL1 Four and a half LIM domains 1 Xq26 2273
FLNC Filamin C, gamma (actin binding protein 280) 7q32-q35 2318
HRC Histidine rich calcium binding protein 19q13.3 3270
MB Myoglobin 22q13.1 4151
MYBPC1 Myosin binding protein C, slow type 12q23.3 4604
MYBPC2 # Myosin binding protein C, fast type 19q13.33 4606
MYL1 Myosin, light polypeptide 1, alkali; skeletal, fast 2q33-q34 4632 MYL2 Myosin, light polypeptide 2, regulatory, cardiac, slow 12q23-q24.3 4633
MYOM2 Myomesin (M-protein) 2, 165kDa 8p23.3 9172
MYOZ1 Myozenin 1 10q22.1 58529
PDLIM3 PDZ and LIM domain 3 4q35 27295
PGAM2 # Phosphoglycerate mutase 2 (muscle) 7p13-p12 5224
PYGM Phosphorylase, glycogen; muscle 11q12-q13.2 5837
ANT1 + Adenine nucleotide translocator of skeletal muscle 1 4q35 291
SLN Sarcolipin 11q22-q23 6588
TCAP Titin-cap (telethonin) 17q12 8557
TNNC2 Troponin C type 2 (fast) 20q12-q13.11 7125
TNNI1 Troponin I type 1 (skeletal, slow) 1q31.3 7135
TNNI2 Troponin I type 2 (skeletal, fast) 11p15.5 7136
TNNT3 Troponin T type 3 (skeletal, fast) 11p15.5 7140
TRDN Triadin 6q22-q23 10345
UQCR Ubiquinol-cytochrome c reductase, 6.4kDa subunit 19p13.3 10975 Genes shown in bold are differentially regulated in OPMD muscle tissue (+ upregulated, # downregulated)
This is further supported by the preliminary results of a gene expression profi ling study on human muscle biopsies comparing healthy individuals with OPMD patients. This study reveals an almost identical expression pattern for the two, with only 4.2% of the transcripts showing differential expression in OPMD muscle.
6.5 Muscle-specifi c formation of intranuclear inclusions in OPMD
With the current knowledge and results presented in this thesis, several theories on the muscle-specifi c initiation of the intranuclear inclusions can be postulated.
6.5.1 Muscle specifi c protein or RNA
To explain the muscle-specifi city of the disease, muscle specifi c proteins or RNAs, in combination with PABPN1 can possibly initiate inclusion formation. In theory this RNA or protein is more highly expressed in muscle than in other tissue, is present in the nucleus, and mainly in post-mitotic cells. A comparison of the gene expression data described in this thesis (Chapter 5) with the preliminary expression data on human OPMD muscle, generates a list of possible candidates having these properties (Table 1).
Further comparison of the two array studies shows that only one gene is differentially expressed in both the cellular model and OPMD muscle (ANT2) but unfortunately it is not present on the list of possible candidates. ANT2 is expressed in myoblasts and functionally it is a part of the mitochondrial permeability transition pore (PTP) which is a translocase for nucleotides (exchange ATP for ADP), forming an important link between energy-producing and energy- consuming processes (45). In humans there are 3 tissue-specifi c ANTs. In differentiated muscle ANT1 is the major representative while ANT2 is mainly expressed in proliferating myoblasts. ANT3 is generally expressed in other tissues, except muscle. Interestingly, ANT1 is in the list of possible candidates as a co-factor for inclusion formation. The muscles affected in OPMD are continuously active and rely on the mitochondria for energy supply. In the theory of inclusion formation dependent on a second factor, ANT1 is higher expressed in the most energy demanding muscles, and if this amount reaches a threshold level (in the presence of mutated PABPN1), inclusions can be formed. The upregulation of ANT2 (normally only expressed in myoblasts) seen in both the cellular model for OPMD (fused myotubes) and in OPMD affected muscle tissue, could thus be a response to a shortage of ANT1, which is trapped in the inclusions. In this theory, entrapment of ANT1 mRNA in the inclusions would be the most logical option because the protein itself is not located in the nucleus. By doing an siRNA experiment against ANT1, results will show if ANT2 is upregulated in reaction to this shortage.
Interestingly in this respect, an OPMD patient has been described (with early onset ptosis and later onset of dysphagia) without a PABPN1 mutation and without INIs. Upon analysis this patient appeared to have a deletion of the 4q35 region in which ANT1 is located (unpublished data, S.M. van der Maarel, Leiden). This supports the hypothesis that a deletion of ANT1 has an identical effect as its entrapment in the INIs, resulting in an OPMD phenotype. This would be a logical pathway affected in OPMD, as mitochondrial changes have been reported earlier in OPMD muscle cells (31-33). This theory would explain why inclusions do not form in myoblasts; ANT1 is very low expressed in these cells. The fact that inclusions can form in non-muscle cellular models overexpressing PABPN1 could be due to a higher expression of other ANT forms in comparison with the in vivo situation. Other cases have been reported with an OPMD phenotype which have no alanine expansion; they also do not show INI formation (46,47). Testing these patients on ANT mutations or deletions will be critical to further assess this theory.
6.5.2 Profi brillar structures that cannot be cleaned up by the proteasome
Another explanation for the inclusion formation is that mutant PAPBN1 forms profi brillar structures which are not recognised as normal by the cell, as is also described in Huntington’s disease (48). These profi brillar structures are bound by chaperones, components of the ubiquitin-proteasome pathway, to be degraded. There is a balance between formation and elimination of profi brilar structures, but at a certain moment inclusions arise because the proteasome cannot keep up. As the onset of OPMD is so fi xed (between 40-50 years), at this particular age other processes may have become more prevalent which also require the proteasome. A possible explanation is that when people grow older, there is a general decrease in muscle mass, with breakdown products also being degraded by the proteasome (49). This might cause proteasomal insuffi ciency for the removal of profi brilar structures and inclusions can form. Among the differentially expressed genes in the OPMD muscle biopsies, the proteasome is also highly upregulated (preliminary data) and proteasome proteins appear
111
to locate to the INIs (50). As the inclusions arise only in post-mitotic cells, it is also possible that part of the profi brillar structures accumulate in time until the ‘inclusion-formation- threshold’ is reached. Gene expression profi ling studies on presymptomatic OPMD patients are essential to provide more insight if the proteasome is already active in cells before intranuclear inclusions exist.
6.5.3 PABPN1-concentration dependent initiation of inclusion formation
The simplest explanation for inclusion formation is that the inclusion formation depends on the concentration of PABPN1 present in the cell. Studies on cellular models of OPMD have shown that overexpression of normal PABPN1 can also cause inclusion formation (Chapter 5) (37). The percentage of inclusion formation is positively correlated with the level of overexpression. This suggests that the concentration of PABPN1 in the cell is tightly regulated under normal circumstances. As an overexpression of PABPN1 is also causing inclusion formation, and in OPMD muscle PABPN1 is twofold higher expressed than in healthy muscle (preliminary results), it is possible that in OPMD the higher amount of PABPN1 present in the cell leads to inclusion formation. In this theory, homozygote OPMD patients would then show an earlier phenotype because they have an even higher overexpression of PABPN1 and reach the critical concentration more rapidly. This would imply that in OPMD PABPN1 may also be overexpressed in other cells, but that the threshold is not reached in vivo. In time however, inclusions can form here too. Homozygotes often suffer from cognitive and behavioural disturbances (51) and in a few heterozygotes neurological changes are reported (52,53) indicating that other cells may also be affected by inclusion formation. Furthermore, Dion et al. generated a transgenic mouse model for OPMD which showed specifi c inclusion formation in neuronal cells (54). Interestingly, the intranuclear inclusions are also detected in neurons from normal rat hypothalamus (55). This would imply that initiation of inclusion formation is not necessarily dependent on muscle-specifi c factors. This also would clarify why several non-muscle cellular models overexpressing normal-length PABPN1 also form intranuclear inclusions; as long as the overexpression reaches a certain threshold, inclusions can form. A remaining question is why the inclusions arise at a certain age. As the inclusions occur only in post-mitotic cells, there could be an accumulation of PABPN1 in these cells, in time eventually reaching the threshold.
6.5.4 Combination of the theories
Questions remain for each of the theories described above. It is possible therefore, that a combination of proteasome dysfunction, abundant expression of a specifi c protein or RNA in muscle and an altered concentration of PABPN1 are all factors involved in the formation of inclusions (Figure 4). Expression profi ling of presymptomatic OPMD muscle and of a timeseries of myoblasts in differentiation (cellular OPMD model) in which the inclusions form in time, may solve this issue.
6.6 Conclusion
This thesis shows that gene expression profi ling is an excellent tool to screen the whole transcriptome in different models for human muscular disease. It provides clues about pathways involved, deregulated pathways and possible leads for treatment. Furthermore, it
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narrows the area of study in such a way, that other (smaller-scale) techniques can be more succesfully applied to answer very precise and specifi c questions.
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