The Role Of Osteocalcin And NAD In Neurodegenerative Diseases

Objectives: The aim of this study is to investigate the effects of osteoblasts-derived osteocalcin on the cognitive dysfunction of Alzheimer's disease(AD)and the important bone metabolism relevant factor ?-Nicotinamide Adenine Dinucleotide(NAD)on the motor dysfunction of Parkinson's disease(PD)and the related mechanisms.Methods: To explore the protective effects of OCN on AD,we used 7-month-old APP/PS1 male mice as AD model.Uncarboxylated osteocalcin(ucOCN,the active form of osteocalcin)was injected intraperitoneally once daily at a fixed time point for 4 weeks with a dose of 1 ug/kg or 10 ug/kg,or intracerebroventricularly with a dose of 20 ng/h for 2 weeks by an osmotic pump implanted subcutaneously under the back and connected to a brain infusion kit.After the intervention,mice were subjected to the open field test(OFT),elevated plus maze test(EPMT)and light-dark transition test(LDTT)to detect the anxiety-like behaviors,and the Morris water maze test(MWMT)to detect the spatial learning and memory ability of the mice.After the behavioral tests were completed,the brain tissues were extracted and immunohistochemical staining for amyloid plaques in hippocampus and cortex,immunofluorescent staining for astrocytes and microglia in hippocampus,and Western Blot for A?-related and AKT/mTOR pathway proteins were performed.In vitro,PC12 cells were used to explore the protective effects of ucOCN against the neurotoxicity of aging A?1-42 and the regulation of ucOCN on AKT/mTOR pathway.To explore the protective effects of NAD on PD,we injected 10 ug 6-OHDA into the right striatum to construct the PD mice model and 20 ug NAD was injected at the same point 4 h before the injection of 6-OHDA.4 weeks after 6-OHDA injection,mice were subjected to the OFT,cylinder test and rotarod test to evaluate the motor function of the mice.After the behavioral tests,the brain tissues were extracted and the immunohistochemical staining for tyrosine hydroxylase(TH)in substantia nigra and striatum and Western Blot for TH and Sirt3-related proteins in striatum were performed.6-OHDA-lesioned PC12 cells were used as PD cell model in vitro and NAD was given 2 h earlier than 6-OHDA lesion to detect its protective role in cell viability,cell morphology,oxidative stress and mitochondrial function.Expressions of Sirt-related proteins in PC12 cells were also detected.Results: Results from the behavioral tests showed that,intraperitoneal injection of ucOCN increased the entries of AD mice into the central area in OFT and the open arm in EPMT and the time into the lit compartment in LDTT.Besides,intraperitoneal injection of ucOCN decreased the average time latency to the platform of AD mice in the training of MWMT and 10 ug/kg ucOCN intervention significantly increased the swimming time of AD mice in the target quadrant in the probe test.Injection of ucOCN into the lateral ventricle also significantly increased the time and entry of AD mice into the center in OFT as well as the time into the open arm in EPMT,but had no significant effect on the performance of AD mice in LDTT.Similarly,intracerebroventricular injection of ucOCN also decreased the time latency of AD mice to the platform from the second day of the training in MWMT and it had a tendency to increase the swimming time in the target quadrant and the platform crossings of AD mice in the probe test.Both the peripheral and central delivery of ucOCN significantly reduced amyloid plaque deposits in hippocampus and cortex,and the APP and A? protein expression in hippocampus.We also observed the inhibition of the glial proliferation in hippocampus after intraperitoneal injection of ucOCN.ucOCN could alleviate the damage of PC12 cell viability induced by aging A?1-42 in vitro,and increase the protein expression of AKT/mTOR both in vitro and in vivo.NAD pre-intervention could improve the use of left forelimb and increase the rotation speed and time latency on the motorized rod of 6-OHDAlesioned mice,but it had no significant improvement in the autonomous movement of the PD mice in the open field test.NAD could also prevent the loss of dopaminergic neurons and fibers in the right substantia nigra and striatum,and increase the expression of TH protein in the right striatum.In vitro,NAD pre-treatment attenuated the PC12 cytotoxicity induced by 6-OHDA,including the decreased cell viability,the damage of cell morphology,the increased ROS level,and the decreased SOD level,mitochondria number,mitochondrial membrane potential and cellular ATP level.NAD could activate the expression of Sirt3 in vitro and in vivo.Conclusions: Osteoblasts-derived OCN could improve the anxiety-like behaviors and spatial learning and memory impairment,alleviate amyloid plaque deposition in hippocampus and cortex,and inhibit the glial hyperproliferation in hippocampus in AD mice,which might be attributed to its activation of the AKT/mTOR pathway.NAD,an important factor in bone metabolism,could alleviate the motor dysfunction and loss of nigrostriatal dopaminergic neurons in PD mice.These protective effects might be exerted via lowering oxidative stress and maintaining mitochondrial homeostasis.Our study adds credence to the beneficial role of osteocalcin and NAD in models of neurodegeneration,provides more evidence for a better understanding of the regulation between bone and brain,and gives rise to the potential of a new treatment strategy for neurodegenerative diseases.