Cigarette smoking has been identified as a major risk factor for osteoporosis decades ago. Several studies have shown a direct relationship between cigarette smoking, decreased bone mineral density, and impaired fracture healing. However, the mechanisms behind impaired fracture healing and cigarette smoking are yet to be elucidated. Migration and osteogenesis of mesenchymal stem/stromal cells (MSCs) into the fracture site play a vital role in the process of fracture healing. In human nicotine, the most pharmacologically active and major addictive component present in tobacco gets rapidly metabolized to the more stable cotinine. This study demonstrates that physiological concentrations of both nicotine and cotinine do not affect the osteogenic differentiation of MSCs. However, cigarette smoke exposure induces oxidative stress by increasing superoxide radicals and reducing intracellular glutathione in MSCs, negatively affecting osteogenic differentiation. Although, not actively producing reactive oxygen species (ROS) nicotine and cotinine inhibit catalase and glutathione reductase activity, contributing to an accumulation of ROS by cigarette smoke exposure. Coincubation with N-acetylcysteine or L-ascorbate improves impaired osteogenesis caused by cigarette smoke exposure by both activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling and scavenging of ROS, which thus might represent therapeutic targets to support fracture healing in smokers.

Hereditary spastic paraplegias (HSPs) comprise a group of genetically heterogeneous neurodegenerative disorders characterized by spastic weakness of the lower extremities. We have generated a Drosophila model for HSP type 10 (SPG10), caused by mutations in KIF5A. KIF5A encodes the heavy chain of kinesin-1, a neuronal microtubule motor. Our results imply that SPG10 is not caused by haploinsufficiency but by the loss of endogenous kinesin-1 function due to a selective dominant-negative action of mutant KIF5A on kinesin-1 complexes. We have not found any evidence for an additional, more generalized toxicity of mutant Kinesin heavy chain (Khc) or the affected kinesin-1 complexes. Ectopic expression of Drosophila Khc carrying a human SPG10-associated mutation (N256S) is sufficient to disturb axonal transport and to induce motoneuron disease in Drosophila. Neurofilaments, which have been recently implicated in SPG10 disease manifestation, are absent in arthropods. Impairments in the transport of kinesin-1 cargos different from neurofilaments are thus sufficient to cause HSP–like pathological changes such as axonal swellings, altered structure and function of synapses, behavioral deficits, and increased mortality.

Several studies have explored the negative effects of cigarette smoke on bone healing; however, the complex pathogenesis still remains unclear. One crucial and primary factor determining effective fracture repair is the recruitment and differentiation of mesenchymal stem cells (MSCs) into bone-forming cells. Recently, primary cilia, microtubule-based sensory organelles, have been shown to be critical in lineage commitment and differentiation of MSCs. Our present study indicates that exposure to cigarette smoke extract (CSE 0.1-10%) impaired osteogenic differentiation of human mesenchymal stem cell line (SCP-1) and interestingly, also affected primary cilia distribution and integrity in these cells during the differentiation. Furthermore, significant amounts of free radicals generated by CSE could be causative of primary cilia loss since treatment with 0.01% of hydrogen peroxide, a prime free radical in CSE, destroyed primary cilia in these cells. The debilitated differentiation of CSE-exposed SCP-1 cells also correlated with the significantly reduced expression of transcription factor and target genes of primary cilia-specific hedgehog signalling, a key player in osteogenic differentiation. As a treatment strategy, co-incubation of the CSE-exposed SCP-1 cells with the antioxidant resveratrol (1 µM) had a protective effect as it significantly reduced free radical production, protected the primary cilia and enhanced osteogenic differentiation. The current study shows for the first time that cigarette smoke affects primary cilia in human MSCs during osteogenic differentiation and treatment with resveratrol could reverse the effects and enhance differentiation, thus opening up potential therapeutic alternatives to treat fracture healing in smokers, in particularly, when delayed fracture healing is assumed.