A Novel Investigation into the Role of Neurotrophic Factors in the Osteocyte and Bone

Degenerative diseases related to ageing are becoming some of the most prevalent health problems experienced by our population. Disorders of the brain and bone such as Alzheimer’s disease (AD), Osteoporosis and Osteoarthritis (OA), respectively, occur in such a large proportion of the ageing population and it is believed that they may be mechanistically linked in their development. This group of studies investigated the idea that proteins locally expressed in the Central Nervous System (CNS) may have roles in the bone and provide a mechanistic insight into the development of disease processes. A number of in vitro models were utilised in order to gain an understanding of the effects that known stimuli of bone resorption and formation (PTH, TNFα and 1,25D) had on APP, APLP-2 and NGF in the osteoblast to osteocyte differentiation process. In vivo studies were also performed to address the effects of App gene knock-out in the long bones of young mice and showed a development related defect in cortical bone remodelling. This study revealed a potential role for APP in the process of bone remodelling, which led onto the investigation of the effects of APP on osteocytes in vitro. There was no obvious effect of soluble APP695 (sAPP695) on osteocyte viability or expression of genes associated with bone resorption or formation. As the ageing population is at high risk of hip fracture and dementia, a screening study of a population of neck of femur fracture (NOF) patients was conducted to determine relationships between the genes of interest and expression of a number of genes involved in the CNS, bone formation and bone resorption as well as skeletal structure. This study provided a novel insight into the NOF cohort as a whole, as well as differences between non-dementia and dementia patients by virtue of their gene expression and skeletal structure of the femur. The overall findings from this chapter suggest a link between AD and osteoporosis through common molecular pathways, which we now believe APP and BACE1, previously referred to only in the context of the brain, to be a part of. These findings give rise to potential alternative treatment strategies for osteoporosis and fracture prevention through the targeting of the novel brain-related proteins. As cleavage of APP leads to the formation of the neurotoxic amyloid beta (Aβ) peptide, a known contributor to the development of AD, it was important to investigate the effects of this peptide on skeletal development through the use of an in vivo model. Utilisation of the AppNL-G-F/NL-G-F familial AD mouse model revealed an accelerated loss of cancellous bone in the 29-week-old male cohort when compares with wild-type littermate control animals, providing strong evidence for roles of Aβ and its precursor APP in the bone maintenance process observed throughout life. It was also shown that these Aβ peptides were present in the bone marrow of the femorae of these mice, which was coupled with an increased number of osteoclasts in these knock-in animals. To further elucidate what the presence of these peptides in the proximity of bone cells could do, human osteocytes were exposed to increasing concentrations of Aβ1-42 peptides in vitro. This cell culture study revealed that osteocyte death could be initiated when exposed to the highest, 15 μM concentration of Aβ1-42 peptides over a 96 hour time course. Furthermore, Aβ1-42 peptides were also capable of upregulating the mRNA expression of markers characteristic of resorption: RANKL, RANKL:OPG and MMP13. This finding, together with the reduction in bone seen with ageing in the mouse model provides a new insight into the possible mechanistic link between Alzheimer’s disease and osteoporosis by virtue of Aβ. The final study conducted was focused around the efficacy of a new potential therapeutic agent for the treatment of pain associated with knee OA. NGF has previously been characterised for its roles in the central nervous system, and in the bone in fracture healing, however its role in other bone pathology such as knee osteoarthritis has not been appreciated previously. Pentosan polysulphate sodium (PPS) was successful in dampening expression of nerve growth factor (NGF) and its pro-protein form (pro-NGF), CNS-related proteins involved in the pain response, in primary human osteocytes exposed to TNFα, a pro-inflammatory cytokine. This study was the first study to demonstrate that osteocytes could express NGF and pro-NGF and that PPS could block the proposed pain response initiated by upregulation of NGF in response to pro-inflammatory stimuli. Overall, the studies conducted in this thesis were designed and implemented to ascertain a new understanding of the current literature about the links between the CNS and bone by virtue of APP, APLP2 and NGF as well as build upon the understanding of the relationship between Alzheimer’s disease and bone loss.