The survival of motor neurons is controlled by multiple factors, which regulate different aspects of their physiology. The identification of these factors is important because of their relevance for motor neuron disease. This thesis began with a search for novel growth factors that naturally keep these neurons alive. Members of the TGF-β superfamily, including Mullerian inhibiting substance (MIS) and bone morphogenetic protein 6 (BMP6), were identified as putative survival factors following a cDNA microarray analysis of a mouse model of motor neuron disease.
MIS is a gonad-derived hormone with a male bias. It induces the degeneration of the female reproductive tract during development and it was thought to have no physiological function outside of the reproductive system. In this thesis, multiple techniques were used to show that adult motor neurons produce MIS and its receptors. The copy number of MIS mRNA in motor neurons was comparable with that of the testis, whereas the mRNA of the MIS type II receptor (MISRII) in motor neurons appeared to be the most abundant receptor of the TGF-β superfamily. These results were confirmed using Western blot and immunohistochemistry. Thus, MIS may exert its function through an autocrine or a paracrine mechanism between neighbouring motor neurons.
The function of MIS was examined using a culture system and a mouse null mutation of MISRII. The in vitro assays showed strong neurotrophic effects of MIS on embryonic motor neurons with the maximum extent of survival being similar to that achieved by the classical motor neuron survival factor, GDNF. MIS has a male bias in utero raising the issue of whether motor neurons are sexually dimorphic. Consistent with this, the number of motor neurons in the lumbar lateral motor column of neonatal male MISRII+/+ mice was 13 % greater than in female mice (P = 0.01). The nuclei of male motor neurons were approximately 20 % larger than their female counterparts (P = 0.000). MISRII-/- male mice had 18 % fewer motor neurons than wild-type males (P = 0.01) and the mean size of their motor neurons was 20 % smaller (P = 0.000). The number and size of motor neurons in the MISRII-/- males was not different to those of MISRII+/+ females. These results implicate MIS as being responsible for neuronal survival as well as producing sexual dimorphism of the limb innervating motor neurons. Since MIS does not appear to be expressed in the embryonic neuromuscular system, it is postulated that MIS is a gonad-derived neurotrophic factor for developing motor neurons.
The BMP type II receptor (BMPRII) was the second most abundant receptor of the TGF-β superfamily expressed by motor neurons. One of its ligands, BMP6, was found to have a neurotrophic effect on motor neurons in culture but was slightly less potent than MIS. BMP6 mRNA was detected in nerve, skeletal muscle and spinal cord, but not in motor neurons. BMP6 immunoreactivity was mainly associated with the myelinated Schwann cells and satellite glia that surround motor neurons. In skeletal muscles, immunoreactivity was not detected in muscle fibers, nor the postsynaptic region of the neuromuscular junction (NMJ). BMP6 was, however, associated with the interstitial cells of skeletal muscles. Double nerve ligations were used to examine whether Schwann cell-derived BMP6 interacts with motor neurons. Consistent with this, BMP6 was retrogradely transported in motor axons. These observations collectively suggest that BMP6 is a glia-derived regulator of motor neurons.
MIS and minority of BMP6 were anterogradely transported towards the NMJ. Their receptors, MISRII and BMPRII, were detected in the postsynaptic portions of the adult NMJ. These observations raised the possibility that MIS and BMP6 may be regulators of the adult NMJ. Since functional redundancy amongst the members of the TGF-β superfamily has been suggested, the function of MIS/BMP6 signaling at the NMJ was therefore examined in mice with muscle-specific deletion of Smad4, a central mediator of TGF-β superfamily pathways. More than 75% of animals lacking Smad4 in muscles died before embryonic day (E) 14 and none survived postnally. This was due to the loss of functional Smad4 in developing cardiac myocytes, which resulted in severe heart defects and early death of embryos. Thus, the function of MIS/BMP6 signaling at the adult NMJ could not be studied.
Finally, this thesis briefly examined the phenotypes of mice carrying double null mutations of MISRII and TGF-β2. The animals died at an early stage and showed a more severe phenotype than either of the single null mutants. This suggests that functional redundancy among members of the TGF-β superfamily exists in many organs.
In summary, motor neurons require multiple sources of growth factors for their survival. MIS and BMP6 were discovered as novel survival factors for motor neurons in this study. MIS was implicated as a regulator of sexual dimorphism in developing motor neurons, whereas both MIS and BMP6 appear to regulate mature motor neurons, and possibly the NMJ.
| Identifer | oai:union.ndltd.org:ADTP/217582 |
| Date | January 2006 |
| Creators | Wang, Pei-Yu, n/a |
| Publisher | University of Otago. Department of Anatomy & Structural Biology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Pei-Yu Wang |
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