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Nerve growth factor R221W responsible for insensitivity to pain is defectively processed and accumulates as proNGF
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Medical and Clinical Genetics.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Medical and Clinical Genetics.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Medical and Clinical Genetics.
Department of Orthopedics, Gällivare Hospital, Gällivare, Sweden.
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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. Vol. 33, no 2, 221-228 p.
Keyword [en]
pain insensitivity, secretion, processing
URN: urn:nbn:se:umu:diva-36994DOI: 10.1016/j.nbd.2008.10.012PubMedID: 19038341OAI: diva2:357003
Available from: 2010-10-14 Created: 2010-10-14 Last updated: 2012-01-20Bibliographically approved
In thesis
1. The use of monogenic disease to study basal and disease associated mechanisms with focus on NGF dependent pain insensitivity and ISCU myopathy
Open this publication in new window or tab >>The use of monogenic disease to study basal and disease associated mechanisms with focus on NGF dependent pain insensitivity and ISCU myopathy
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.
Umeå University medical dissertations, ISSN 0346-6612 ; 1463
monogenic, disease, NGF, receptor, pain insensitivity, ISCU, myopathy, splicing
National Category
Other Basic Medicine
Research subject
Molecular Medicine
urn:nbn:se:umu:diva-51140 (URN)978-91-7459-326-6 (ISBN)
Public defence
2012-02-10, Betula, By 6M, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Available from: 2012-01-20 Created: 2012-01-11 Last updated: 2012-01-20Bibliographically approved

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