title: Mechanism of myostatin action during satellite cell activation and muscle wasting.

authors: McFarlane, Craig Desmond

subjects: Myostatin

abstract: Myostatin, a Transforming Growth Factor-beta (TGF-β) superfamily member, has been well characterised as a negative regulator of muscle growth and development.  In support, inactivation or mutation of the myostatin gene results in a dramatic increase in skeletal muscle mass, however excess myostatin inhibits myogenesis.  Recently, myostatin has also been shown to have a role in post-natal muscle growth.  Myostatin regulates activation, proliferation and self-renewal of the muscle satellite cell pool.  Moreover, loss of myostatin results in enhanced skeletal muscle regeneration in response to injury, whereas increased post-natal myostatin expression is associated with many skeletal muscle wasting conditions.  Furthermore, myostatin has been shown to directly induce cachexia following subcutaneous injection of Myostatin over-expressing cells into mice.  Despite studies implicating myostatin in the regulation of post-natal skeletal muscle growth, little is known about the processes through which myostatin activity is regulated or the mechanisms through which myostatin functions.  Thus this thesis examines regulation of myostatin activity through proteolytic processing, and signaling mechanisms through which myostatin acts to regulate the satellite cell pool and to promote skeletal muscle proteolysis.  
In this thesis it is demonstrated that processing and secretion of Myostatin is relatively reduced in differentiated myotubes as compared to proliferating myoblasts.  Furthermore, processing of Myostatin is developmentally regulated, with decreased Myostatin processing occurring during foetal muscle development when compared to post-natal adult muscle.  It is also demonstrated that mature Myostatin negatively regulates furin promoter activity.  Furin protease is critical for the processing of several members of the TGF-β superfamily, thus a mechanism is proposed whereby myostatin negatively auto-regulates its proteolytic processing during development to facilitate the process of myoblast differentiation.  
It was further demonstrated that over-expression of Pax7 in C3H10T1/2 multipotent cells enhances myogenic conversion in these cells.  However, over-expression of Pax7 in C2C12 myoblasts delays the onset of differentiation, concomitant with an increase in the population of quiescent, satellite cell-like reserve cells.  Furthermore, treatment with Myostatin down-regulates Pax7 expression, while Pax7 expression was higher in myostatin-null myoblasts as compared to wild-type myoblast cultures.  Furthermore, absence of myostatin alters cell heterogeneity, whereby an increase in Pax7+/MyoD- reserve cell populations is observed.  Pax7 expression persists longer through differentiation in cultured primary myoblasts from myostatin-null animals when compared to wild-type counterparts.  Reserve cell populations were also measured, and consistent with increased expression of Pax7, there is an increased pool of quiescent  self-renewed  reserve cells in differentiated cultures from myostatin-null mice as compared with wild-type cultures.  Taken together, these results suggest that increased expression of Pax7 regulates the self-renewal process of satellite cells, and furthermore, growth factors such as myostatin signal through Pax7 to regulate the self-renewed pool of satellite cells. 
In this thesis it is shown that myostatin induces cachexia through a mechanism independent of NF-κB.  Myostatin treatment results in a cachexia phenotype with a reduction in myotube number and size in vitro, as well as a loss of body mass in vivo.  Furthermore, the expression of the myogenic genes myoD and pax3 are reduced, while NF-κB localisation and expression remains unchanged.  Expression of the ubiquitin-associated genes Atrogin-1, MuRF-1 and E214k are shown to be up-regulated following Myostatin treatment.  The mechanism behind myostatin-mediated cachexia was further investigated.  It is shown that myostatin antagonises the IGF-1/PI3-K/AKT hypertrophy pathway by inhibiting AKT phosphorylation, thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes.  
In addition, microarray analysis resulted in the identification of a potentially interesting downstream target gene of myostatin during the induction of cachexia.  Initial characterisation of CXXC5, or MM1 as it was renamed, was also performed.  Over-expression of MM1 in vitro results in the up-regulation of components of the ubiquitin-proteasome pathway including Atrogin-1, E214k, E220k and RC2.  It was further demonstrated that increased expression of MM1 enhances the level of protein ubiquitin-conjugation.  Furthermore, over-expression of MM1 results in myotube collapse and the formation of multinucleated myosacs.  It was also demonstrated that the MM1-induced myotube collapse results in disruption of the typical myotube microtubule structure.  Therefore, these data suggest that MM1 is a muscle wasting-inducing gene which functions in the regulation of myostatin-mediated cachexia.  
Therefore data presented in this thesis highlights a mechanism through which myostatin is regulated, and further delineates the role of myostatin in controlling several key processes during post-natal myogenesis, namely satellite cell replenishment and skeletal muscle wasting.

text: Mechanism of myostatin action during satellite cell activation and muscle wasting. Myostatin, a Transforming Growth Factor-beta (TGF-β) superfamily member, has been well characterised as a negative regulator of muscle growth and development.  In support, inactivation or mutation of the myostatin gene results in a dramatic increase in skeletal muscle mass, however excess myostatin inhibits myogenesis.  Recently, myostatin has also been shown to have a role in post-natal muscle growth.  Myostatin regulates activation, proliferation and self-renewal of the muscle satellite cell pool.  Moreover, loss of myostatin results in enhanced skeletal muscle regeneration in response to injury, whereas increased post-natal myostatin expression is associated with many skeletal muscle wasting conditions.  Furthermore, myostatin has been shown to directly induce cachexia following subcutaneous injection of Myostatin over-expressing cells into mice.  Despite studies implicating myostatin in the regulation of post-natal skeletal muscle growth, little is known about the processes through which myostatin activity is regulated or the mechanisms through which myostatin functions.  Thus this thesis examines regulation of myostatin activity through proteolytic processing, and signaling mechanisms through which myostatin acts to regulate the satellite cell pool and to promote skeletal muscle proteolysis.  
In this thesis it is demonstrated that processing and secretion of Myostatin is relatively reduced in differentiated myotubes as compared to proliferating myoblasts.  Furthermore, processing of Myostatin is developmentally regulated, with decreased Myostatin processing occurring during foetal muscle development when compared to post-natal adult muscle.  It is also demonstrated that mature Myostatin negatively regulates furin promoter activity.  Furin protease is critical for the processing of several members of the TGF-β superfamily, thus a mechanism is proposed whereby myostatin negatively auto-regulates its proteolytic processing during development to facilitate the process of myoblast differentiation.  
It was further demonstrated that over-expression of Pax7 in C3H10T1/2 multipotent cells enhances myogenic conversion in these cells.  However, over-expression of Pax7 in C2C12 myoblasts delays the onset of differentiation, concomitant with an increase in the population of quiescent, satellite cell-like reserve cells.  Furthermore, treatment with Myostatin down-regulates Pax7 expression, while Pax7 expression was higher in myostatin-null myoblasts as compared to wild-type myoblast cultures.  Furthermore, absence of myostatin alters cell heterogeneity, whereby an increase in Pax7+/MyoD- reserve cell populations is observed.  Pax7 expression persists longer through differentiation in cultured primary myoblasts from myostatin-null animals when compared to wild-type counterparts.  Reserve cell populations were also measured, and consistent with increased expression of Pax7, there is an increased pool of quiescent  self-renewed  reserve cells in differentiated cultures from myostatin-null mice as compared with wild-type cultures.  Taken together, these results suggest that increased expression of Pax7 regulates the self-renewal process of satellite cells, and furthermore, growth factors such as myostatin signal through Pax7 to regulate the self-renewed pool of satellite cells. 
In this thesis it is shown that myostatin induces cachexia through a mechanism independent of NF-κB.  Myostatin treatment results in a cachexia phenotype with a reduction in myotube number and size in vitro, as well as a loss of body mass in vivo.  Furthermore, the expression of the myogenic genes myoD and pax3 are reduced, while NF-κB localisation and expression remains unchanged.  Expression of the ubiquitin-associated genes Atrogin-1, MuRF-1 and E214k are shown to be up-regulated following Myostatin treatment.  The mechanism behind myostatin-mediated cachexia was further investigated.  It is shown that myostatin antagonises the IGF-1/PI3-K/AKT hypertrophy pathway by inhibiting AKT phosphorylation, thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes.  
In addition, microarray analysis resulted in the identification of a potentially interesting downstream target gene of myostatin during the induction of cachexia.  Initial characterisation of CXXC5, or MM1 as it was renamed, was also performed.  Over-expression of MM1 in vitro results in the up-regulation of components of the ubiquitin-proteasome pathway including Atrogin-1, E214k, E220k and RC2.  It was further demonstrated that increased expression of MM1 enhances the level of protein ubiquitin-conjugation.  Furthermore, over-expression of MM1 results in myotube collapse and the formation of multinucleated myosacs.  It was also demonstrated that the MM1-induced myotube collapse results in disruption of the typical myotube microtubule structure.  Therefore, these data suggest that MM1 is a muscle wasting-inducing gene which functions in the regulation of myostatin-mediated cachexia.  
Therefore data presented in this thesis highlights a mechanism through which myostatin is regulated, and further delineates the role of myostatin in controlling several key processes during post-natal myogenesis, namely satellite cell replenishment and skeletal muscle wasting.

year: 2007

title: Development of immunoaffinity columns and immunoassays for the collection, partial purification and measurement of sporidesmin A and its metabolites

authors: Briggs, Lyn R.

abstract: Monoclonal and polyclonal antibodies were produced for the development of competitive enzyme-linked immunosorbent assays (cELISA) for sporidesmin A, the mycotoxin that causes facial eczema in grazing livestock. Derivatives of sporidesmin conjugated to a number of proteins were used as immunising antigens and plate-coating antigens for cELISA. Two immunoassays, each detecting the presence of a different region of the sporidesmin A molecule have been developed. They allow the two major sporidesmins found in grass or in Pithomyces chartarum cultures to be distinguished. Organic solvent extraction of samples was avoided so that water-soluble metabolites could also be detected.

To assist in the selection of animals resistant to facial eczema, the cELISAs were applied to a study of the metabolism of sporidesmin A in sheep. The cELISAs distinguish between sporidesmin A and its metabolites, as one antibody detects only sporidesmin A and the other detects sporidesmin A and metabolites. After sheep were dosed with sporidesmin A it was possible to monitor the excretion of the toxin in bile and its metabolite(s) in both bile and urine using the appropriate cELISA. There were no differences between resistant and susceptible sheep in the cumulative totals or excretion rates of immunoreactive metabolite in animals dosed with sporidesmin A.

The detection of sporidesmin metabolites in sheep urine by cELISA was shown to have potential as a biomarker for measuring exposure to toxic pastures which could give early warning of the risk of facial eczema.

An immunoaffinity chromatography matrix was prepared by coupling antibody to cyanogen bromide-activated Sepharose. The matrix bound sporidesmin A and immunoreactive metabolites from urine, and bound analytes were eluted with 40% methanol in water. The matrix capacity for sporidesmin A was 900 ng of sporidesmin A bound per mL of matrix, and for sporidesmin metabolites from urine it was approximately 500 ng of sporidesmin A immunoreactive equivalents per mL of matrix.

A method was developed for the preparation of milk for immunoaffinity chromatography so that blocking of columns by milk fats was avoided. Sample concentration obtained using the immunoaffinity chromatography lowered the limit of quantification for the previously developed cELISA for sporidesmin A in milk by a factor of 100.

A trial was carried out to determine whether sporidesmin residues appeared in milk following experimental ingestion of the toxin. Using the cELISA, sporidesmin metabolites were detected in milk samples collected after dosing. Further evidence for the presence of immunoreactive material was obtained by using immunoaffinity chromatography to recover the material from milk. Positive results in the sporidesmin A specific cELISA suggest the presence of free sporidesmin A at picogram levels. Purification from a much larger sample, and analyses by HPLC and mass spectrometry are needed to confirm this result.

It should now be possible to use the anti-sporidesmin antibodies in immunochemical technologies and formats which are suitable for on-farm use. These could be used to help minimise exposure to sporidesmin A and provide an important advance in animal health.

text: Development of immunoaffinity columns and immunoassays for the collection, partial purification and measurement of sporidesmin A and its metabolites Monoclonal and polyclonal antibodies were produced for the development of competitive enzyme-linked immunosorbent assays (cELISA) for sporidesmin A, the mycotoxin that causes facial eczema in grazing livestock. Derivatives of sporidesmin conjugated to a number of proteins were used as immunising antigens and plate-coating antigens for cELISA. Two immunoassays, each detecting the presence of a different region of the sporidesmin A molecule have been developed. They allow the two major sporidesmins found in grass or in Pithomyces chartarum cultures to be distinguished. Organic solvent extraction of samples was avoided so that water-soluble metabolites could also be detected.

To assist in the selection of animals resistant to facial eczema, the cELISAs were applied to a study of the metabolism of sporidesmin A in sheep. The cELISAs distinguish between sporidesmin A and its metabolites, as one antibody detects only sporidesmin A and the other detects sporidesmin A and metabolites. After sheep were dosed with sporidesmin A it was possible to monitor the excretion of the toxin in bile and its metabolite(s) in both bile and urine using the appropriate cELISA. There were no differences between resistant and susceptible sheep in the cumulative totals or excretion rates of immunoreactive metabolite in animals dosed with sporidesmin A.

The detection of sporidesmin metabolites in sheep urine by cELISA was shown to have potential as a biomarker for measuring exposure to toxic pastures which could give early warning of the risk of facial eczema.

An immunoaffinity chromatography matrix was prepared by coupling antibody to cyanogen bromide-activated Sepharose. The matrix bound sporidesmin A and immunoreactive metabolites from urine, and bound analytes were eluted with 40% methanol in water. The matrix capacity for sporidesmin A was 900 ng of sporidesmin A bound per mL of matrix, and for sporidesmin metabolites from urine it was approximately 500 ng of sporidesmin A immunoreactive equivalents per mL of matrix.

A method was developed for the preparation of milk for immunoaffinity chromatography so that blocking of columns by milk fats was avoided. Sample concentration obtained using the immunoaffinity chromatography lowered the limit of quantification for the previously developed cELISA for sporidesmin A in milk by a factor of 100.

A trial was carried out to determine whether sporidesmin residues appeared in milk following experimental ingestion of the toxin. Using the cELISA, sporidesmin metabolites were detected in milk samples collected after dosing. Further evidence for the presence of immunoreactive material was obtained by using immunoaffinity chromatography to recover the material from milk. Positive results in the sporidesmin A specific cELISA suggest the presence of free sporidesmin A at picogram levels. Purification from a much larger sample, and analyses by HPLC and mass spectrometry are needed to confirm this result.

It should now be possible to use the anti-sporidesmin antibodies in immunochemical technologies and formats which are suitable for on-farm use. These could be used to help minimise exposure to sporidesmin A and provide an important advance in animal health.

year: 1997

title: Media psychology, symbolic power and social justice in Aotearoa

authors: Hodgetts, Darrin

subjects: media

abstract: Psychologists reside in a world saturated by media. We work in professional contexts where guidelines for practice foreground ethical obligations to address issues of social justice. This paper addresses both these contextual dimensions of psychological research and practice. We explore the social significance of increased media production by Maori in challenging the tendency in mainstream media to marginalize Maori concerns while promoting Pakeha perspectives. The analysis focuses on the recent 'Inside Out documentary - Hikoi', which was initiated by two young Maori women as a challenge to media framing of Maori protests as 'unjustified' and 'disruptive' acts. We illustrate how this documentary furthers public dialogue regarding the foreshore and seabed controversy by promoting an alternative depiction of a Maori protest, which emphasize the history of grievances and social unity. The implications of such representations for psychologists working to address issues of social justice and to challenge abuses of symbolic power are discussed.

text: Media psychology, symbolic power and social justice in Aotearoa Psychologists reside in a world saturated by media. We work in professional contexts where guidelines for practice foreground ethical obligations to address issues of social justice. This paper addresses both these contextual dimensions of psychological research and practice. We explore the social significance of increased media production by Maori in challenging the tendency in mainstream media to marginalize Maori concerns while promoting Pakeha perspectives. The analysis focuses on the recent 'Inside Out documentary - Hikoi', which was initiated by two young Maori women as a challenge to media framing of Maori protests as 'unjustified' and 'disruptive' acts. We illustrate how this documentary furthers public dialogue regarding the foreshore and seabed controversy by promoting an alternative depiction of a Maori protest, which emphasize the history of grievances and social unity. The implications of such representations for psychologists working to address issues of social justice and to challenge abuses of symbolic power are discussed.

year: 2004-11-19