| Study purpose:Parkinson's disease (PD) is a severe degenerative disorder that is characterized by massive dopaminergic neuronal loss in substantia nigra (SN). The pathogenesis of PD or mechanism of neuronal degeneration is still largely obscure. The p75 neurotrophin receptor (p75NTR), one key member of tumor necrosis receptor superfamily, has a low-affinity binding to mature neurotrophins, i.e. nerve growth factor (NGF), brain-derived neurotrophic factor and neurotrophin-3, and mediate neuronal cell survival, differentiation and neurite outgrowth. Rencent evidence has shown that p75NTR can in high affinity bind to the precursor of neurotrohpins such as pro-NGF, and trigger neuronal cell apoptosis by assistance of sortilin, a co-receptor of p75NTR. A line of evidence has shown that p75NTR plays important roles in controlling cell apoptosis in the developing or injury states of brain. While expression of p75NTR was upregulated in pathological condiations in the adult such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD), its abnormal upregulation is recognized to involve in neuronal degeneration in disease onset and progression of these neurodegenerative diseases.However, it is still not known if p75NTR is also involved in pathogenesis of PD. By focusing on this question in this study, cellular localization of p75NTR and its co-receptor sortilin were first identified in the substantia nigra neurons. By using kainic acid (KA)-lesioned animal model, upregulated expression of p75NTR and sortilin was then examined, and their roles in inducing neuronal degeneration and triggering intracellular apoptosis signaling were investigated. The results of this study have provided important evidence to reveal significance of p75NTR and sortilin apoptotic signalling in disease progression and new intervention target for treatment of PD.Material and Methods:①KA-lesioned animal models were prepared by stereotaxic injection of KA into substantia nigra of adult rats.②Reverse transcript polymerase chain reaction (RT-PCR) was quantitatively performed to examine expression and levels of p75NTR, sortilin, and proNGF mRNAs in the substantia nigra of control and KA-lesioned rats.③Immunohistochemistry, double immunofluorescence and laser scanning confocal microscopy were used to localize p75NTR, sortilin, proNGF in the dopaminergic neurons in the substantia nigra.④Western Blotting was quantitatively performed to confirm expression and levels of p75NTR, sortilin, proNGF proteins in the substantia nigra.⑤Fluoro-Jade C (FJC), a specific stain for degenerating neurons, was carried out to visualizing neuronal degeneration in the substantia nigra of KA-lesioned rats.⑥Analysis of intracellular apoptosis signaling was done to check activation of JNK, AIF signaling pathway in the substantia nigra neurons in KA-lesioned rats.Main results:1. Identification of p75NTR, sortilin expression in the dopaminergic neurons and their upregulation in the substantia nigra after KA insult①Expression of p75NTR and sortilin in dopaminergic neurons was detected by using immunocytochemistry. In the midbrain sections, p75NTR or sortilin-positive neurons were observed, and they were mainly distributed in the substantia nigra pars compacta. Double immunofluorescence and laser scanning confocal microscopy confirmed that 47% TH-positive neurons expressed p75NTR, and sortilin was expressed in all TH-positive neuronal ones.②Expression of p75NTR and sortilin mRNAs was further found in the substantia nigra by RT-PCR experiments. Results confirmed PCR bands of p75NTR, or sortilin mRNA in expected 309bp, or 438bp respectively.③Up-regulation of p75NTR and sortilin was found in the substantia nigra of KA-lesioned animal model. Relative optical density of p75NTR increased from 0.489±0.079 at control to 1.06±0.11 at KA-lesion (p<0.01, t test), and sortilin increased from 0.724±0.056 at control to 1.226±0.078 at KA-lesion (p<0.01, t test). Expression of p75NTR increased into all TH-positive neurons, and sortilin-positive neurons increased from 204±4 to 293±13 in KA-lesion rats (p﹤0.01). 2.Up-regulation of p75NTR and sortilin expression induced degeneration of the dopaminergic neuron in the substnatia nigra after KA insult①Colocalization of p75NTR and sortilin was confirmed in the substantia nigra neurons. Taken together with finding on p75NTR/TH, sortlin/TH double labeled neurons, it clearly appeared that p75NTR/sortilin was most colocalized in the dopaminergic neurons in the substantia nigra pars compacta, and these p75NTR/sortilin neurons constitute about 71.8% of total p75NTR ones.②Expression of proNGF was further found in TH-positive neurons and GFAP-positive astrocytes in the substantia nigra. These proNGF/TH neurons were mostly distributed in the ventral tier of substantia nigra pars compacta, and proNGF/GFAP astrcytes were seen in the KA-lesion state.③Up-regulated expression of proNGF was found in KA-lesioned substantia nigra which was in consistent with upregulation of p75NTR and sortilin. Results showed that relative optical density of proNGF mRNA increased from 0.18±0.037 at control to 0.703±0.059 at KA-lesion (p <0.01, t test). Cell counts indiacted that proNGF/TH neurons increased from 40±5 at control to 75±5 at KA-lesion (p﹤0.01).④Quantitative and dynamic analysis were made on p75NTR, proNGF, sortilin, FJC, or TH-positive cells at time-points of d1, d3 and d7 afte KA-lesion. The results showed that p75NTR, sortilin, proNGF-positive cells increased gradually, but TH-positive neurons decreased, and at the same time FJC-positive cells increased. The relationship between p75NTR and TH, sortilin and TH, proNGF and TH were negative correlation, correlation coefficient were -0.988, -0.998, -0.993, respectively. The increase of FJC and decrease of TH was negative correlation, the correlation coefficient was -0.999.3. Upregulation of p75NTR and sortilin expression might triger intracellular apoptotic JNK and AIF signals in KA-lesion state①Activation of JNK signaling pathway was found in KA-lesion substantia nigra. It is well known that c-jun belongs to capase-dependent signaling pathway. The results showed that expression of c-jun and phosphorylation levels of c-jun increased dramatically in the substantia nigra after KA-lesion, c-jun/TH double labeled neurons increased from 10±1 at control to 20±2 at KA-lesion. Western blotting confirmed that relative density of c-jun protein increased from 0.749±0.048 at control to1.087±0.109 at KA-lesion (p<0.05). Besides, the p-c-jun/TH neurons increased from 26±2 at control to 65±4 at KA-lesion (p<0.01).②Activation of AIF signal was also detected in the substantia nigra after KA-lesion. Evidence has shown that AIF belongs to capase-independent signaling pathway. This result indicated that AIF expression was significantly up-regulated in KA-lesioned substantia nigra. In the nigra region, AIF-positve increased from 195±3 at control to 243±14 at KA-lesion. On the other hand, AIF/TH positive neurons decreased from 177±4 at control to 112±4 at KA-lesion, indicating some AIF/TH neurons was died after KA-insult.Preliminary conclusion①Expression of p75NTR, sortilin was first identified in dopaminergic neurons of substantia nigra in this study, and upregulated levels were found in KA injury state.②Up-regulation of p75NTR and sortilin appeared to induce degenerative death of dopaminergic neurons of the substantia nigra in KA-lesioned animal model.③Up-regulated p75NTR and sortilin might be involved in activation of intracellular JNK and AIF signaling in substantia nigra of KA-lesioned rats.These data has provided evidence that p75NTR, sortilin apoptotic signalling may be involved in inducing degeneration of dopaminergic neuron in the substantia nigra. This study also suggested that p75NTR may play important role in pathogenesis or disease progression of PD and represent new intervention target for treatment of PD.
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