In this study, increased Apo D released from astrocytes was observed in PD models. When co-cultured with astrocytes, due to the increased of astrocytic Apo D, it could significantly alleviate the lesion of DAergic neurons with MPP+ treatment. Increased Apo D levels was regulated by the phosphorylation at Y99 of TAp73 in astrocytes, thus astrocytes-specific knockdown of TAp73 could exacerbate the neurodegeneration in PD. Furthermore, exogenous Apo D exerted neuroprotective effects against MPTP/MPP+ toxicity via anti-oxidation and anti-apoptosis. Our findings reported that astrocytic-derived Apo D was essential for DAergic neuronal survival in PD, which might provide new therapeutic targets for PD (Fig. 8).
Contrary to most apolipoproteins, Apo D mRNA expression in humans is found in several tissues including the CNS, mammary glands, spleen, adipose tissues, adrenals, and skin25,31,40. Recently, clinical studies reported that elevated levels of Apo D in the plasma were correlated significantly with PD stage, indicating that Apo D might be a valid marker for the progression of PD22. In contrast to humans, Apo D mRNA expression in mice and rats is mainly restricted to the CNS25,41–44.25,41–44 The role of Apo D and its effects in the CNS has long been underestimated, despite the fact that Apo D is highly elevated during aging and neural injury.45 In this study, we observed that the levels of Apo D were significantly up-regulated in the SN in MPTP-treated PD mice. In addition, the up-regulated Apo D levels is accompanied by the increased GFAP in the SN in MPTP-treated mice, which is in line with the fact that astrocytes are major site of Apo D production in the CNS27,46,47. The neurotoxin MPTP can lead to severe damage to the nigrostriatal DAergic system in mice48,49. All data available support the idea that pretreatment with Apo D significantly prevented MPTP-induced dyskinesia, alleviated the reduction in the number of nigral DAergic neurons and promoted the synthesis and release of DA in Str. Our results also observed that exogenous Apo D applied to MES23.5 cells could exert neuroprotective effects in MPP+-induced neurotoxicity. Furthermore, our findings indicated that the mice with conditional knockdown of Apo D in astrocytes in the SN exhibited exacerbated PD pathology. All data available support that astrocytic-derived Apo D were essential for DAergic neuronal survival in PD, which might provide new therapeutic targets for PD.
Mitochondrial dysfunction and oxidation stress-mediated apoptosis play an important role in the pathogenesis of PD50,51. The decrease of mitochondrial potential induces the release of cytochrome C (Cyt C) into the cytoplasm, activates caspase-3 and induces apoptosis52. Bcl-2 inhibits caspase-3-dependent apoptosis by binding to apoptosis-promoting Bax protein, and its ratio is a key factor in determining whether cells are apoptotic53,54. In the present study, we observed that astrocytic Apo D inhibited the apoptosis of DAergic neurons via attenuating the accumulation of ROS and regulating the expression of Bcl-2 and Bax in MPP+-treated DAergic neurons. These neuroprotective and antioxidant roles of Apo D might be closely associated with its capacity of reducing radical-propagating lipid hydroperoxides by three methionine (Met) residues (Met49, Met93, and Met157) 55.
Neuron-astrocyte crosstalk is critical for brain metabolism and neuron protection against oxidative stress in diverse neuropathological disorders56,57.56,57 In PD, astrocytes have protective effects on the development and regeneration of the nigrostriatal DAergic system and protect neurons from oxidative stress induced by reactive oxygen species (ROS) 9,58,59. The activation of signaling cascades by neurotrophic factors such as glial-derived GDNF could induce neuro-restoration in rodent PD models60,61. It is widely supported by gene profile studies62,63 that mature neurons do not express Apo D mRNA. Instead, neurons can uptake Apo D from the extracellular environment30. However, Apo D can transfer from astrocytes to neurons by extracellular vesicles, which is an underlying mechanism of the neuroprotective effect of Apo D32. In our study, we observed that the increased astrocytic Apo D in co-culture system of primary cultured VM neurons and astrocytes directly protect the cell viability of DAergic neurons treated with MPP+, which uncovered that the protective effect of Apo D via astrocyte-neuron crosstalk could be a common pathway for neuroprotection and self-repair in PD.
In this regard, it is important to explore the mechanisms underlying the up-regulation of Apo D levels and its release in PD. Previous studies showed that Apo D is a direct transcriptional target of the p53 family member genes and found that the expression of Apo D was specifically up-regulated by TAp7364. TAp73 protects against aging by regulating mitochondrial activity and preventing ROS accumulation. TAp73-KO mice show increased ROS production and oxidative stress sensitivity64. We observed the levels of Apo D and TAp73 were significantly increased in MPP+-treated astrocytes. Moreover, the protein levels of Apo D were significantly reduced in TAp73 knockdown astrocytes and conditional knockdown of TAp73 on astrocytes in MPTP-treated PD mice, suggesting that TAp73 was essential for the increased astrocytic Apo D levels and its release in PD.
Unlike those of the p53 gene, the mutations in the p73 gene are very rare in tumors, suggesting that the decrease in TAp73 activity and expression detected in those tumors are caused mainly by coordinated post-translational modifications of TAp7365, including the ubiquitin-dependent proteasomal degradation pathway, phosphorylation, acetylation, and small ubiquitin-related modifier (SUMOylation)65. The phosphorylation of TAp73 through its interaction with kinase is considered an essential pathway for the regulation of TAp73 activity and stability under normal conditions and in response to genotoxic stress37–39. Several studies have reported that TAp73 is phosphorylated at Tyr99 by the non-receptor tyrosine kinase c-Abl in response to DNA damage, resulting in an increase in TAp73 stability and pro-apoptotic activity37,38,66. Our study demonstrated that increased levels of TAp73 and its phosphorylation at Y99 in astrocytes were required for the increased Apo D levels and its release. These observations gave us a hint that phosphorylation at Y99 of TAp73 might play a key role in regulating astrocytic Apo D maintaining brain homeostasis in PD. The deep and exact mechanism is worthy of further investigation.
While Apo D is up-regulated approximately 2- to 3-fold in neurodegenerative disease or aging, its dynamic range of expression appears to be far greater67. Of note, although Apo D can be efficiently produced as a functional recombinant protein in microbial host cells, its therapeutic value for long-term systemic application is restricted by the blood-brain barrier68. In our study, we observed that MPTP/MPP+ could induced the increased levels of astrocytic Apo D and appear to produce favorable effects in the treatment of PD. Furthermore, the levels of Apo D and its release from astrocytes in PD models were regulated by TAp73. In this regard, enhancing the levels of astrocytic Apo D over an extended period of time might have visible clinical benefits in PD therapy and could possibly even delay the process of neurodegeneration.