Caffeic acid reduces A53T α-synuclein by activating JNK/Bcl-2-mediated autophagy in vitro and improves behaviour and protects dopaminergic neurons in a mouse model of Parkinson's disease.

The human A53T mutant of α-synuclein tends to aggregate and leads to neurotoxicity in familial Parkinson's disease (PD). The aggregation of α-synuclein is also found in sporadic PD. Thus, targeting α-synuclein clearance could be used as a drug-discovery strategy for PD treatment. Caffeic acid (CA) has shown neuroprotection in Alzheimer's disease or cerebral ischaemia; however, it is unclear whether CA confers neuroprotection in α-synuclein-induced PD models. Here we focus on whether and how A53T α-synuclein is affected by CA. We assessed the effect of CA on cell viability in SH-SY5Y cells overexpressing A53T α-synuclein. Pathway-related inhibitors were used to identify the autophagy mechanisms. Seven-month-old A53T α-synuclein transgenic mice (A53T Tg mice) received CA daily for eight consecutive weeks. Behaviour tests including the buried food pellet test, the pole test, the Rotarod test, open field analysis, and gait analysis were used to evaluate the neuroprotective effect of CA. Tyrosine hydroxylase and α-synuclein were assessed by immunohistochemistry or western blot in the substantia nigra (SN). We found that CA alleviated the cell damage induced by overexpressing A53T α-synuclein and that CA reduced A53T α-synuclein by activating the JNK/Bcl-2-mediated autophagy pathway. The efficacy of CA on A53T α-synuclein degradation was reversed by the autophagy inhibitor bafilomycin A1 and the JNK inhibitor SP600125. In A53T Tg mice, CA improved behavioural impairments, attenuated loss of dopaminergic neurons, enhanced autophagy and reduced α-synuclein in the SN. Thus, the results provide scientific evidence for the neuroprotective effect of CA in PD. Our work lays the foundation for CA clinical trials to treat PD in the future.

Release of soluble and vesicular purine nucleoside phosphorylase from rat astrocytes and microglia induced by pro-inflammatory stimulation with extracellular ATP via P2X7 receptors.

Purine nucleoside phosphorylase (PNP), a crucial enzyme in purine metabolism which converts ribonucleosides into purine bases, has mainly been found inside glial cells. Since we recently demonstrated that PNP is released from rat C6 glioma cells, we then wondered whether this occurs in normal brain cells. Using rat primary cultures of microglia, astrocytes and cerebellar granule neurons, we found that in basal condition all these cells constitutively released a metabolically active PNP with Km values very similar to those measured in C6 glioma cells. However, the enzyme expression/release was greater in microglia or astrocytes that in neurons. Moreover, we exposed primary brain cell cultures to pro-inflammatory agents such as lipopolysaccharide (LPS) or ATP alone or in combination. LPS alone caused an increased interleukin-1ß (IL-1ß) secretion mainly from microglia and no modification in the PNP release, even from neurons in which it enhanced cell death. In contrast, ATP administered alone to glial cells at high micromolar concentrations significantly stimulated the release of PNP within 1 h, an effect not modified by LPS presence, whereas IL-1ß secretion was stimulated by ATP only in cells primed for 2 h with LPS. In both cases ATP effect was mediated by P2X7 receptor (P2X7R), since it was mimicked by cell exposure to Bz-ATP, an agonist of P2X7R, and blocked by cell pre-treatment with the P2X7R antagonist A438079. Interestingly, ATP-induced PNP release from glial cells partly occurred through the secretion of lysosomal vesicles in the extracellular medium. Thus, during inflammatory cerebral events PNP secretion promoted by extracellular ATP accumulation might concur to control extracellular purine signals. Further studies could elucidate whether, in these conditions, a consensual activity of enzymes downstream of PNP in the purine metabolic cascade avoids accumulation of extracellular purine bases that might concur to brain injury by unusual formation of reactive oxygen species.