Two back-to-back studies published in the July 23rd issue of Cell, one from Columbia University Medical Center and the other from Johns Hopkins researchers, further the hypothesis that metabolic control and bone remodelling are inextricably linked. Both studies point to osteocalcin, a hormone released by bone, as a key mediator of this link.
The Johns Hopkins study used a conditional knock-out in mice to specifically suppress the expression of the insulin receptor in osteoblasts, the bone-forming cells of the skeletal system. As the mutant mice aged they became fat, had elevated blood sugar, and were glucose intolerant and resistant to insulin, mirroring the picture of diabetes in humans. The researchers found that the mutant mice had fewer osteoblasts, reduced bone formation and lower levels of circulating undercarboxylated osteocalcin (the active form of the hormone). The study showed that signalling via the insulin receptor in osteoblasts suppressed Twist2, an inhibitor of osteoblast development, and enhanced expression of osteocalcin, a mediator of insulin sensitivity and secretion.
The Columbia study links the complete bone remodelling process to energy regulation. Osteocalcin is released from osteoblasts predominantly in an inactive, carboxylated form. The researchers demonstrated that insulin signalling in osteoblasts stimulates release of inactive osteocalcin and activates osteoclasts, which activate the osteocalcin via decarboxylation in a bone-resorption-dependent manner.
The studies clearly have potential impact on human therapy, although significant questions remain. As yet the receptor for undercarboxylated osteocalcin is unknown, so the mechanism by with the hormone stimulates insulin release is unclear. Further work will be necessary to understand the interplay between skeletal- and metabolic-homeostasis in humans.
Wnt signalling plays an important role in the development and maintenance of many organs and tissues, and appears to be especially important in regulating bone mass. Enhanced Wnt signalling has the potential to speed up healing and mice genetically modified to have prolonged Wnt signalling heal more quickly than control mice. So far it hasn’t been possible to directly test the effect of administering Wnt ligands because of difficulties in purifying and formulating the proteins, but researchers at Stanford University School of Medicine have now solved the problem by packaging the proteins in liposomal vesicles.
When Wnt3a-loaded liposomes were administered to mice with bone injury, within 3 days the animals had 3.5 times more new bone growth than animals that received no treatment or animals that received Wnt protein without the carrier liposomes. After 4 weeks, the bone had completely healed in the animals treated with Wnt-carrying liposomes whereas untreated animals took another 2 weeks. Wnt was shown to act by increasing the proliferation of bone progenitor cells – Wnt-responsive cells are found on the inner surface of the bone and participate in normal bone maintenance and in bone growth in response to injury. A liposomal formulation would also be suitable for use in people and could, after much further evaluation, be used to speed bone healing. Members of the bone morphogenetic protein family are currently used in spinal fusions and to treat some fractures but can cause bone to grow in the wrong place.
In some animals, such as flatworms and zebrafish, the Wnt pathway allows tissue regeneration without scarring and the authors hope that their study might also provide the basis for new treatments for stroke and heart attack where scar tissue formed as part of the normal healing process impairs later function.
Photo credit: NASA Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is one of the most common neuromuscular diseases worldwide and attacks the neurons responsible for controlling voluntary muscles. Few treatment options are currently available for ALS patients but researchers at UT Southwestern Medical Center and Harvard University have shown that microRNA-206 (miR-206) plays a crucial role in the progression of ALS. miRNAs are small non-coding RNA molecules which down-regulate gene expression and dysfunction of miRNAs has been associated with a number of diseases. In ALS, as the affected neurons stop signalling to muscle cells, the muscles atrophy. Although the skeletal muscles attempt to reinnervate themselves by signalling to healthy neurons via miRNA-206, eventually the surviving neurons are unable to cope, the muscle cells die and the ability of the brain to control voluntary movement is lost.
Mice that are genetically deficient in miRNA-206 form normal neuromuscular synapses during development but, in the ALS mouse model, disease progression is faster in mice that are deficient in miRNA-206. miRNA-206 is required for efficient regeneration of neuromuscular synapses after acute nerve injury and is dramatically induced in the mouse model of ALS. The effects of miRNA-206 in slowing ALS were suggested to be mediated, at least in part, through histone deacetylase 4 and fibroblast growth factor signalling pathways. miRagen Therapeutics hope to exploit the newly discovered role for miRNA-206 in neuromuscular maintenance to develop treatments for patients suffering from ALS and other neuromuscular diseases. Because miR-206 is only produced by skeletal muscles, such treatments may have a limited risk of side effects.
Duchenne muscular dystrophy (DMD), the most prevalent of more than twenty types of genetic disorder that result in progressive muscle weakness, is caused by a mutation in the gene that encodes the protein, dystrophin. Dystrophin forms one subunit of a glycoprotein complex that acts as a structural scaffold linking the extracellular matrix to the intracellular cytoplasm. Without dystrophin, the muscle fibre membranes become damaged by the large mechanical forces experienced by contractile tissues and the muscle fibres eventually die. Since the gene for dystrophin is on the X chromosome, only boys are affected and, although the disorder is predominantly one of muscle degeneration, some boys also have learning or behavioural difficulties.
Mice which have a loss-of-function mutation in the dystrophin gene (mdx mice) are used as a model for human DMD but new research led by scientists at King’s College London and funded by the Muscular Dystrophy Campaign suggests that these animals may not be suitable for studying the neurological effects of human DMD. The study looked at the genes for other proteins that make up the dystrophin glycoprotein complex and found significant differences between mice and humans. The main heterodimeric partner of dystrophin at the heart of the glycoprotein complex is α-dystrobrevin, which can exist in a substantial number of isoforms as a result of complex transcriptional and post-transcriptional regulation. The different isoforms of α-dystrobrevin influence the recruitment of other proteins into the dystrophin glycoprotein complex, leading to variations in structure and function. The researchers found that mouse, rat and hamster brains have fewer than half the number of α-dystrobrevin isoforms found in the brains of most other mammals (including humans), suggesting that there are likely to be fundamental differences between the dystrophin glycoprotein complexes of mice and humans, and calling into question the current use of mice to model neurological aspects of human DMD. Guinea pigs appear to be more similar to humans in terms of α-dystrobrevin isoforms and may provide a more suitable model.
Spinal Muscular Atrophy (SMA) describes a group of diseases where motor neurons of the spinal cord and brain stem, which are critical for stimulation of muscle cells, degenerate and die. Lacking the appropriate input, the muscle cells become much smaller (atrophy) and patients display symptoms of muscle weakness. Affected muscles are those involved in voluntary movement and patients may have difficulty swallowing, breathing, crawling, walking and with head/neck movement. SMA is an autosomal recessive genetic disease and for a child to be affected both parents must be carriers of the abnormal gene and both must pass this gene on to their child. The incidence of SMA is estimated at 1 in 6000 births and this condition is responsible for the death of more infants than any other genetic disease.
SMA results when the SMN1 (survival of motor neuron 1) gene, which encodes survival of motor neuron (SMN) protein, is missing or mutated. SMN is critical to the survival and health of motor neurons. The closely related survival of motor neuron SMN2 gene is retained in all SMA patients but does not produce sufficient SMN protein to prevent the development of clinical symptoms. Although SMN2 differs from SMN1 by only a single nucleotide, the change affects the efficiency with which exon 7 is incorporated into the mRNA transcript. As a result, SMN2 produces less full-length mRNA and protein than SMN1.
In 2001, researchers at Ohio State University showed that aclarubicin was able to restore levels of SMN in a mouse model by altering the incorporation of exon 7 into SMN2 transcripts. Although aclarubicin is too toxic to consider for development, the work prompted scientists at Paratek Pharmaceuticals to screen related tetracycline analogues. This has now resulted in the identification of PTK-SMA1, a synthetic tetracycline-like compound, as a lead candidate. PTK-SMA1, like aclarubicin, increases levels of SMN by correcting SMN2 splicing. The study, conducted in collaboration with scientists at Cold Spring Harbor and Rosalind Franklin Univeristy, is published in Science Translational Medicine.
Further collaborative research to progress the program to IND filing is being supported by a five-year, multi-million dollar cooperative agreement from the National Institute of Neurological Disorders and Stroke (NINDS) and by the Families of SMA funding program.