Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: The levels of osteocalcin (OCN) in the serum or cerebrospinal fluid (CSF) of control and 6-hydroxydopamine (6-OHDA)-induced Parkinson’s disease (PD) rat models. The CSF OCN levels in PD rats dramatically decreased compared to control rats ( A , n = 17). No difference in the serum OCN levels was found between control and PD rats ( B , n = 19). Expression of GPR158 and GPRC6A in various brain-derived tissues of 8-week-old rats ( C , n = 3). Coimmunoprecipitation of 8-week-old mice striata and proteins were then subjected to western blot analysis using anti-Gpr158 and anti-GPRC6a antibodies ( D , n = 3). Values were obtained from three independent experiments and results are given as the mean ± SEM. * P < 0.05.

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Control, Expressing, Derivative Assay, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: Effect of intraperitoneal (i.p.) injection of OCN on distance traveled (A) and rearing responses (B) in the open-field test (OFT). The cylinder test (C) and elevated body swing test (EBST; D ) were used to evaluate locomotor asymmetry. Compared with the sham groups, 6-OHDA injection decreased the distance traveled by rats in the OFT, and reduced the use of contralateral (left) forelimb in the cylinder test. The injection of 4 μg/kg OCN significantly improved the movement distance of 6-OHDA-lesioned rats in the 4th week ( P < 0.001), while 40 μg/kg OCN increased the number of rearing responses in the 3rd week ( P < 0.05). The representative traces of the OFT are shown in the lower panel of (A,B) . In the cylinder test, 4 μg/kg OCN improved the use of the contralateral forelimb ( P < 0.05). No obvious differences were observed in the EBST. n = 8–10, * P < 0.05 (compared with the 6-OHDA group).

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Injection

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: Effect of intra-striatum injection of OCN on distance traveled (A) , rearing responses (B) in OFT. The cylinder test (C) and EBST (D) were used to evaluate locomotor asymmetry. Compared with the sham groups, 6-OHDA injection decreased the distance traveled by rats, while the injection of OCN (4 μg) or nerve growth factor (NGF) significantly improved movement distance (A) , but not rearing impairment (B) of 6-OHDA-lesioned rats in OFT. The representative traces of the OFT are shown in the lower panel of (B) . No obvious differences were observed in the cylinder test (C) or the EBST (D) . n = 9–11, * P < 0.05 (compared with the 6-OHDA group).

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Injection

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: The protective effect of OCN on tyrosine hydroxylase (TH + ) neurons and fibers in the nigrostriatal system in the unilateral 6-OHDA-lesioned PD rat model. 6-OHDA induced ~90% dopaminergic neuronal loss on the lesioned side compared to the intact side of the substantia nigra (SN), which was improved by i.p. OCN injection (A) . Similarly, the decrease in TH-positive cell number in the SN of the lesioned side was also significantly alleviated by OCN injection (B) . The results from western blots measuring TH were consistent with immunohistochemistry in the striatum (C) . In the intra-striatum injection OCN group, 6-OHDA induced ~50% dopaminergic neuronal loss in the lesioned side compared to the intact side of the SN, which was improved by OCN injection (D) . Similarly, the decrease in TH-positive cell number in the SN of lesioned side was also significantly alleviated by intra-striatum OCN injection (E) . The results from western blots measuring TH were consistent with immunohistochemistry in the striatum (F) . n = 16, * P < 0.05 (compared with the 6-OHDA group).

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Injection, Western Blot, Immunohistochemistry

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: The effect of OCN on the proliferation of astrocytes and microglia. 6-OHDA significantly increased the number of GFAP-positive astrocytes in the lesioned side compared to the intact side both in the SN (A) and striatum (B) , which was inhibited by OCN injection. The same function of OCN on microglial proliferation was also observed, which was stained with Iba1 (C,D) . n = 16. Green = TH, Red = GFAP or Iba1, Blue = Nucleus.

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Injection, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: Neuroprotective effects of OCN on 6-OHDA-induced neurotoxicity. PC12 cells were incubated with different concentrations of 6-OHDA for 24 h, and cell viability was measured by cell counting kit-8 (CCK-8; A ). Pretreatment of PC12 cells with various concentrations of OCN (1–100 ng/ml) was performed 2 h before exposure to 100 μM 6-OHDA, and the cell viability was measured at 24 h (B) . The effects of OCN on the auto-oxidation of 6-OHDA were measured spectrophotometrically at 490 nm using a microplate reader (C) . The results from western blots showed that treatment with 100 μM 6-OHDA increased the expression of caspase-3 and decreased the expression of bcl-2, and both changes were reversed by pre-incubation with OCN (D) . 6-OHDA decreased the expression of phosphorylated AKT (Ser473) and phosphorylated GSK3β (Ser9) at 24 h (E) , and both reductions were reversed by pretreatment with OCN (F) . Similarly, 6-OHDA injection into the right striatum also resulted in decreased expression of p-AKT and p-GSK3β, and both reductions were inhibited by direct injection of OCN into the striatum (G) . Values were obtained from three independent experiments. * P < 0.05 (compared with the control group), # P < 0.05 (compared with the 6-OHDA group).

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Incubation, Cell Counting, CCK-8 Assay, Western Blot, Expressing, Injection, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Osteocalcin Ameliorates Motor Dysfunction in a 6-Hydroxydopamine-Induced Parkinson’s Disease Rat Model Through AKT/GSK3β Signaling

doi: 10.3389/fnmol.2018.00343

Figure Lengend Snippet: OCN protects PC12 cells from 6-OHDA-induced neurotoxicity. Apoptotic cells were measured by flow cytometry (A–E) . The number of apoptotic cells was counted in the R2 (AV + /7-amino-actinomycin, 7-AAD + , the late phase apoptotic cells) and R4 (AV + /7-AAD − , the early phase apoptotic cells) regions, and the apoptotic rate was calculated (F) . The CCK-8 test showed that OCN could attenuate the neuronal toxicity induced by 6-OHDA through activating AKT/GSK3β, while LY294002 reversed the neuroprotective effect of OCN on PC12 cells (G) through inhibiting the phosphorylation of AKT and GSK3β (H) . Values were obtained from three independent experiments. * P < 0.05 (compared with the control group), # P < 0.05 (compared with the 6-OHDA group).

Article Snippet: A rat OCN ELISA kit was purchased from MyBioSource (San Diego, CA, USA, Catalog: MBS2022619).

Techniques: Flow Cytometry, CCK-8 Assay, Phospho-proteomics, Control