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The use of monogenic disease to study basal and disease associated mechanisms with focus on NGF dependent pain insensitivity and ISCU myopathy
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Medical and Clinical Genetics.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Monogenic diseases make excellent models for the study of gene functions and basal cellular mechanisms in humans. The aim of this thesis was to elucidate how genetic mutations affect the basal cellular mechanisms in the monogenic diseases Nerve growth factor (NGF) dependent pain insensitivity and Iron-Sulphur cluster assembly protein U (ISCU) myopathy.

NGF dependent pain insensitivity is a rare genetic disorder with clinical manifestations that include insensitivity to deep pain, development of Charcot joints, and impaired temperature sensation but with no effect on mental abilities. The disease is caused by a missense mutation in the NGFβ gene causing a drastic amino acid substitution (R221W) in a well-conserved region of the protein. NGF is secreted in limited amounts by its target tissues and is important for the development and maintenance of the cholinergic forebrain neurons as well as the sensory and sympathetic neurons. To reveal the underlying mechanisms of disease we performed functional studies of the mutant NGF protein. We could show that mutant NGF was unable to induce differentiation of PC12 cells as a consequence of impaired secretion. Furthermore, mutant NGF had different intracellular localisation compared to normal NGF and resided mostly in its unprocessed form proNGF. Mature NGF and proNGF have different binding properties to the receptors TrkA and p75. Individuals with mutations in TRKA are, aside from pain insensitive mentally affected; therefore it has been proposed that the R221W mutation mainly affects the interaction with p75. In agreement with this, we could show that R221W NGF was able to bind and activate TrkA whereas the interaction with p75 was impaired as compared to normal NGF.

ISCU myopathy is a monogenic disease where the affected patients suffer from severe exercise intolerance resulting in muscle cramps and sometimes severe lactic acidosis. The disease is caused by a point mutation in the last intron of the Iron sulphur cluster assembly gene, ISCU, resulting in the inclusion of a part of the intron in the mRNA. ISCU functions as a scaffold protein in the assembly of iron-sulphur (Fe-S) clusters important for electron transport in Kreb’s cycle and the respiratory chain. We have shown that ISCU is vital in mammals since complete knock-down of Iscu in mice results in early embryonic death. The deletion of ISCU homologous in lower organisms has also been shown fatal. In spite this central role in energy metabolism the disease is restricted to the patient’s skeletal muscles while other energy demanding organs seem unaffected. To address this contradiction we examined if tissue-specific differences in the splicing of mutant ISCU could explain the muscle-specific phenotype. We could show that the splicing pattern did, indeed, differ with more incorrectly spliced ISCU in muscle compared to other tissues. This was accompanied by a decrease in Fe-S containing proteins in muscle, while no decrease was observed in other tissues. Alternative splicing is more common then previously thought and may depend upon interacting factors and/or differences in the surrounding milieu. To reveal plausible mechanisms involved in the tissue-specific splicing we identified nuclear factors that interacted with the region where the mutation was located. Five interacting factors were identified, out of which three affected the splicing of ISCU. PTBP1 was shown to repress the incorrect splicing while IGF2BP1 and RBM39 repressed the formation of normal transcript and could also counteract the effect of PTBP1. IGF2BP1 was the only factor that showed higher affinity to the mutant sequence making it a possible key factor in the incorrect splicing of the mutant ISCU gene.

Together, these results offer important insights into the cellular mechanisms causing these diseases. We found impaired secretion and inaccurate sorting of NGF to be cellular mechanisms contributing to NGF dependent pain insensitivity while tissue-specific splicing of ISCU was found to be the event contributing to the phenotype of ISCU myopathy.

Place, publisher, year, edition, pages
Umeå: Umea university, Department of Medical Biosciences , 2012. , 46 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1463
Keyword [en]
monogenic, disease, NGF, receptor, pain insensitivity, ISCU, myopathy, splicing
National Category
Other Basic Medicine
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:umu:diva-51140ISBN: 978-91-7459-326-6 (print)OAI: oai:DiVA.org:umu-51140DiVA: diva2:479043
Public defence
2012-02-10, Betula, By 6M, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2012-01-20 Created: 2012-01-11 Last updated: 2012-01-20Bibliographically approved
List of papers
1. Nerve growth factor R221W responsible for insensitivity to pain is defectively processed and accumulates as proNGF
Open this publication in new window or tab >>Nerve growth factor R221W responsible for insensitivity to pain is defectively processed and accumulates as proNGF
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2009 (English)In: Neurobiology of Disease, ISSN 0969-9961, E-ISSN 1095-953X, Vol. 33, no 2, 221-228 p.Article in journal (Refereed) Published
Abstract [en]

We have previously identified a homozygous missense (R221W) mutation in the NGFB gene in patients with loss of deep pain perception. NGF is important not only for the survival of sensory neurons but also for the sympathetic neurons and cholinergic neurons of the basal forebrain; however, it is the sensory neurons that are mainly affected in patients with mutant NGFB. In this report, we describe the effects of the mutation on the function of NGF protein and the molecular mechanisms that may underlie the pain insensitivity phenotype in these patients. We show that the mutant NGF has lost its ability to mediate differentiation of PC12 cells into a neuron-like phenotype. We also show that the inability of PC12 cells to differentiate is due to a markedly reduced secretion of mature R221W NGF. The R221W NGF is found mainly as proNGF, in contrast to wild-type NGF which is predominantly in the mature form in both undifferentiated and differentiated PC12 cells. The reduction in numbers of sensory fibers observed in the patients is therefore probably due to loss of trophic support as a result of drastically reduced secretion of NGF from the target organs. Taken together, these data show a clear decrease in the availability of mutant mature NGF and also an accumulation of proNGF in both neuronal and non-neuronal cells. The differential loss of NGF-dependent neurons in these patients, mainly affecting sensory neurons, may depend on differences in the roles of mature NGF and proNGF in different cells and tissues.

Place, publisher, year, edition, pages
San Diego: Academic P., 2009
Keyword
pain insensitivity, secretion, processing
Identifiers
urn:nbn:se:umu:diva-36994 (URN)10.1016/j.nbd.2008.10.012 (DOI)19038341 (PubMedID)
Available from: 2010-10-14 Created: 2010-10-14 Last updated: 2017-12-12Bibliographically approved
2. Purification and Characterization of the Nerve Growth Factor R221W mutant causing Insensitivity to Pain
Open this publication in new window or tab >>Purification and Characterization of the Nerve Growth Factor R221W mutant causing Insensitivity to Pain
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

 

We have previously identified a homozygous missense (R221W) mutation in the NGFβ gene which causes insensitivity to pain in patients. The mutation impairs the secretion of NGF and the majority of the protein accumulates as proNGF. NGF mediates its function by binding and activating the TrkA and p75 receptors and is important for the survival of the sensory and sympathetic neurons as well as the cholinergic neurons of the basal forebrain. However, the R221W mutation seems to discriminate between these types of neurons as it is the sensory neurons that are mainly affected in the patients. A second human NGFβ mutation causes a more severe form of pain insensitivity with additional anhidrosis and cognitive dysfunctions in affected patients which is also seen in patients with mutations in the gene encoding the TrkA receptor. Because R221W NGF cause a less severe phenotype we hypothesised that the mutation mainly affects the p75 interaction which is also strengthened by the fact that the substitution is located in a region known to interact with p75. In this report, we show that R221W NGF is able to bind and activate TrkA at a level comparable to wild-type NGF in cells stably expressing TrkA while the activation of the downstream target ERK1/2 is impaired in cells that co-express TrkA and p75. We also describe the effects of the mutation in terms of expression and purification properties from E.coli which indicate the likelihood that eukaryotic folding machinery is needed for correct folding of R221W NGF.

Keyword
NGF, TrkA, p75, Pain insensitivity, protein
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:umu:diva-51117 (URN)
Available from: 2012-01-11 Created: 2012-01-11 Last updated: 2012-01-20
3. Tissue-specific splicing of ISCU results in a skeletal muscle phenotype in myopathy with lactic acidosis, while complete loss of ISCU results in early embryonic death in mice
Open this publication in new window or tab >>Tissue-specific splicing of ISCU results in a skeletal muscle phenotype in myopathy with lactic acidosis, while complete loss of ISCU results in early embryonic death in mice
2011 (English)In: Human Genetics, ISSN 0340-6717, E-ISSN 1432-1203, Vol. 129, no 4, 371-378 p.Article in journal (Refereed) Published
Abstract [en]

Hereditary myopathy with lactic acidosis (HML) is caused by an intron mutation in the iron-sulphur cluster assembly gene (ISCU) leading to incorporation of intron sequence into the mRNA. This results in a deficiency of Fe-S cluster proteins, affecting the TCA cycle and the respiratory chain. The proteins involved in the Fe-S machinery are evolutionary conserved and shown to be fundamental in all organisms examined. ISCU is expressed at high levels in numerous tissues in mammals, including high metabolic tissues like the heart, suggesting that a drastic mutation in the ISCU gene would be damaging to all energy-demanding organs. In spite of this, the symptoms in patients with HML are restricted to skeletal muscle, and it has been proposed that splicing events may contribute to the muscle specificity. In this study we confirm that a striking difference in the splicing pattern of mutant ISCU exists between different tissues. The highest level of incorrectly spliced ISCU mRNA was found in skeletal muscle, while the normal splice form predominated in patient heart. The splicing differences were also reflected at a functional level, where loss of Fe-S cluster carrying enzymes and accumulation of iron were present in muscle, but absent in other tissues. We also show that complete loss of ISCU in mice results in early embryonic death. The mice data confirm a fundamental role for ISCU in mammals and further support tissue-specific splicing as the major mechanism limiting the phenotype to skeletal muscle in HML.

Keyword
iron-sulfur proteins; succinate-dehydrogenase; paroxysmal myoglobinuria; deficiency; exercise; clusters; mutation; mitochondria; metabolism; maturation
National Category
Medical Genetics
Research subject
Medicine
Identifiers
urn:nbn:se:umu:diva-40798 (URN)10.1007/s00439-010-0931-3 (DOI)000289275200002 ()21165651 (PubMedID)
Available from: 2011-03-09 Created: 2011-03-09 Last updated: 2017-12-11Bibliographically approved
4. The defective splicing caused by the ISCU intron mutation in patients with myopathy with lactic acidosis is repressed by PTBP1 but can be de-repressed by IGF2BP1
Open this publication in new window or tab >>The defective splicing caused by the ISCU intron mutation in patients with myopathy with lactic acidosis is repressed by PTBP1 but can be de-repressed by IGF2BP1
2012 (English)In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 33, no 3, 467-470 p.Article in journal (Refereed) Published
Abstract [en]

Hereditary myopathy with lactic acidosis (HML) is caused by an intron mutation in the iron-sulfur cluster assembly gene ISCU which leads to the activation of cryptic splice sites and the retention of part of intron 4. This incorrect splicing is more pronounced in muscle than in other tissues, resulting in a muscle-specific phenotype. In this study, we identified five nuclear factors that interact with the sequence harboring the mutation and analyzed their effect on the splicing of the ISCU gene. The identification revealed three splicing factors, SFRS14, RBM39 and PTBP1, and two additional RNA binding factors, matrin 3 (MATR3) and IGF2BP1. IGF2BP1 showed a preference for the mutant sequence, whereas the other factors showed similar affinity for both sequences. PTBP1 was found to repress the defective splicing of ISCU, resulting in a drastic loss of mutant transcripts. In contrast, IGF2BP1 and RBM39 shifted the splicing ratio toward the incorrect splice form.

Keyword
ISCU;hereditary myopathy;alternative splicing;PTBP1
National Category
Basic Medicine
Identifiers
urn:nbn:se:umu:diva-50593 (URN)10.1002/humu.22002 (DOI)
Available from: 2012-01-02 Created: 2011-12-14 Last updated: 2017-12-08Bibliographically approved

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