(M-498) Sympathetic Neuron Precursor Hyperactivity Mediates Bone Loss in Model of Alzheimer’s Disease

Introduction::
Alzheimer’s disease (AD) patients are at a significantly increased risk of osteoporosis relative to similarly aged individuals, suggesting an interplay between AD pathology and bone loss. Clinical studies have shown a consistent increase in the serum levels of key inflammatory cytokines such as tumor necrosis factor alpha (TNF-⍺), interleukin-1 beta (IL-1β), and the pleiotropic IL-6 in these disorders [1],[2]. A link between AD and osteoporosis is further supported by the dysregulation of the sympathetic nervous system (SNS) – which heavily innervates bone [3] – in AD pathology [4]. Specifically, abnormal SNS signaling has been associated with bone formation/resorption cycle disruption. However, the degree to which AD-related SNS dysregulation impacts osteoporosis development is poorly understood. Toward exploring these pathologies, we performed a preliminary, longitudinal study of a TNF-⍺-stimulated co-culture model of poly(ethylene glycol) diacrylate (PEGDA) hydrogel-encapsulated sympathetic neuron (SN)-like rat neuroendocrine pheochromocytoma-12 (PC12) cells and human bone marrow-derived mesenchymal stem cells (hMSCs), for investigating osteoporosis in a chronically inflamed state. Following confirmation that abnormally activated SN-like PC12 cells induced alterations in hMSC osteogenesis, we assessed whether AD patient-derived cholinergic sympathetic neuroblasts (CSNs) and MSCs would demonstrate inherent differences in sympathetic activation state and hMSC osteogenesis relative to healthy controls by implementing human induced pluripotent stem cells (iPSCs).







Materials and Methods::
For the initial PC12 study, PC12 cells were activated with 50 ng/mL of recombinant murine nerve growth factor (NGF) for 72 hours prior to suspension in PEGDA to induce a hyperactive, pathological state. Encapsulated PC12s and MSCs – at a concentration of 5 × 10⁶ cells/mL – were seeded in the luminal and subluminal chambers, respectively, and the hydrogels were allowed to cure under UV exposure for 6 minutes. Designated groups received a combined medium containing 50 ng/mL of recombinant human bone morphogenic protein 2 (rhBMP-2), with or without 10 ng/mL of TNF-⍺. After 2 weeks of culture, samples were analyzed by multiplex immunoassays to assess the expression of markers associated with osteo-regulation, including vascular endothelial growth factor (VEGF), osteopontin (OPN), receptor activator of nuclear factor kappa-B ligand (RANKL), and type 1 collagen alpha 1 (COL1A1). For the iPSC-based study, differentiations of MSCs and SNs from iPSCs were performed based on a modified version of both the manufacturer’s protocol (STEMCELL) and procedures stated by Frith and Tsakiridis [5], respectively. To induce an AD-analogous inflammatory disease state, cultures were subjected to a cytokine cocktail mix of 5 ng/mL TNF-⍺, 10 ng/mL IL-6, and 5 ng/mL IL-1β, in addition to 50 ngl/mL rhBMP-2 for 7 days. Endpoint analysis was performed using immunocytochemistry and multiplex immunoassay assessing neuronal health.






Results, Conclusions, and Discussions::
Exposure of MSC monocultures to proinflammatory TNF-⍺ resulted in a significant decrease in VEGF and OPN (~1.8-fold and ~2.3-fold, with p=0.0003 and p=0.03, respectively), two markers critical to bone regeneration and formation [6, 7], shown in Figure 1A. RANKL – a protein associated with osteoclastogenesis [8] – was similarly increased ~8.7-fold (p=0.0000014) in the TNF-⍺ treated group relative to untreated controls. These results suggest that the MSCs are moving away from osteoblastic differentiation, putatively driving a pathological shift toward bone reformation. In the presence of activated PC12s (Figure 1B), MSC behavior was differentially affected. Notably, COL1A1 expression was significantly decreased (~2.1-fold, p=0.03), and OPN was further reduced (~7.7-fold, p=0.000034) compared to TNF-⍺ stimulated MSC monocultures. COL1A1 expression is associated with the production of COL1, a principal constituent found in bone [9]. Overall, these results indicate that osteogenesis is markedly decreased in a chronically inflamed state by reducing bone formation and regeneration while potentially increasing bone resorption. This study provides inaugural evidence that inflammation-induced sympathetic hyperactivity in AD potentiates bone loss. Our current investigation into iPSC-derived CSNs and MSCs builds upon these results, suggesting a potential detrimental role of the AD pathological state in osteoporosis.






Acknowledgements (Optional): : This work was supported by National Institute of Health Research Grant R03AG067140 to M.A.G. and M.S.H and R03AG067970 to M.S.H.




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