Parkinson’s disease (PD) is the second most common neurodegenerative disease worldwide, being largely characterized by motor features. MicroRNAs (miRNAs) are small non-coding RNAs, whose deregulation has been associated with neurodegeneration in PD. In this study, miRNAs targeting cell death and/or inflammation pathways were selected and their expression compared in the serum of PD patients and healthy controls. We used two independent cohorts (discovery and validation) of 20 idiopathic PD patients (iPD) and 20 healthy controls each. We also analyzed an additional group of 45 patients with a mutation in the leucine-rich repeat kinase 2 (LRRK2) gene (LRRK2-PD). miRNA expression was determined using Taqman qRT-PCR and their performance to discriminate between groups was assessed by receiver operating characteristic (ROC) curve analysis. We found miR-146a, miR-335-3p, and miR-335-5p downregulated in iPD and LRRK2-PD patients versus controls in both cohorts. In addition, miR-155 was upregulated in LRRK2-PD compared to iPD patients showing an appropriate value of area under the ROC curve (AUC 0.80) to discriminate between the two groups. In conclusion, our study identified a panel of inflammatory related miRNAs differentially expressed between PD patients and healthy controls that highlight key pathophysiological processes and may contribute to improve disease diagnosis.

Parkinson’s disease (PD) is driven by dopaminergic neurodegeneration in the substantia nigra pars compacta (SN) and striatum. Although apoptosis is considered the main neurodegenerative mechanism, other cell death pathways may be involved. In this regard, necroptosis is a regulated form of cell death dependent on receptor interacting protein 3 (RIP3), a protein also implicated in apoptosis and inflammation independently of its pro-necroptotic activity. Here, we explored the role of RIP3 genetic deletion in in vivo and in vitro PD models. Firstly, wild-type (Wt) and RIP3 knockout (RIP3ko) mice were injected intraperitoneally with MPTP (40 mg/kg, i.p.), and sacrificed after either 6 or 30 days. RIP3ko protected from dopaminergic neurodegeneration in the SN of MPTP-injected mice, but this effect was independent of necroptosis. In keeping with this, necrostatin-1s (10 mg/kg/day, i.p.) did not afford full neuroprotection. Moreover, MPTP led to DNA fragmentation, caspase-3 activation, lipid peroxidation and BAX expression in Wt mice, in the absence of caspase-8 cleavage, suggesting intrinsic apoptosis. This was mimicked in primary cortical neuronal cultures exposed to the active MPTP metabolite. RIP3 deficiency in cultured cells and in mouse brain abrogated all phenotypes. Curiously, astrogliosis was increased in the striatum of MPTP-injected Wt mice and further exacerbated in RIP3ko mice. This was accompanied by absence of microgliosis and reposition of glial cell line-derived neurotrophic factor (GDNF) levels in the striata of MPTP-injected RIP3ko mice when compared to MPTP-injected Wt mice, which in turn showed a massive GDNF decrease. RIP3ko primary mixed glial cultures also presented decreased expression of inflammation-related genes upon inflammatory stimulation. These findings hint at possible undescribed non-necroptotic roles for RIP3 in inflammation and MPTP-driven cell death, which can contribute to PD progression.

Necroptosis is a regulated form of necrosis, which may be critical in the pathogenesis of neurodegenerative diseases. Neuroinflammation, characterized by the activation of glial cells such as microglia, is closely linked with neurodegenerative pathways and constitutes a major mechanism of neural damage and disease progression. Importantly, inhibition of necroptosis results in disease improvement, unveiling an alternative approach for therapeutic intervention. In the present study, we screened a small library of new molecules, potentially inhibitors of necroptosis, using two cellular models of necroptosis. A new oxazolone, Oxa12, reduced tumour necrosis factor α (TNF-α)-induced necroptosis in mouse L929 fibrosarcoma cells. Notably, Oxa12 strongly inhibited zVAD-fmk-induced necroptosis in murine BV2 microglial cells. Moreover, Oxa12 blocked phosphorylation of mixed-lineage kinase domain-like protein (MLKL), and interfered with necrosome complex formation, indicating that Oxa12 targets components upstream of MLKL. In fact, in silico molecular docking studies revealed that Oxa12 is occupying a region similar to the 1-aminoisoquinoline type II kinase inhibitor inside the receptor-interacting protein 1 (RIP1) kinase domain. Finally, in microglial cells, Oxa12 attenuated zVAD-fmk- and lipopolysaccharide (LPS)-induced inflammatory processes, as revealed by a marked decrease of TNF-α and/or IL-1β expression. More specifically, Oxa12 negatively targeted c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways, as well as NF-κB activation. Overall, we identified a strong lead inhibitor of necroptosis that is also effective at reducing inflammation-associated events. Oxa12 is a promising candidate molecule for further development to target disease states dependent on RIP kinase activity.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, mainly characterized by motor deficits correlated with progressive dopaminergic neuronal loss in the substantia nigra pars compacta (SN). Necroptosis is a caspase-independent form of regulated cell death mediated by the concerted action of receptor-interacting protein 3 (RIP3) and the pseudokinase mixed lineage domain-like protein (MLKL). It is also usually dependent on RIP1 kinase activity, influenced by further cellular clues. Importantly, necroptosis appears to be strongly linked to several neurodegenerative diseases, including PD. Here, we aimed at identifying novel chemical inhibitors of necroptosis in a PD-mimicking model, by conducting a two-step screening. Firstly, we phenotypically screened a library of 31 small molecules using a cellular model of necroptosis and, thereafter, the hit compound effect was validated in vivo in a sub-acute 1-methyl-1-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) PD-related mouse model. From the initial compounds, we identified one hit—Oxa12—that strongly inhibited necroptosis induced by the pan-caspase inhibitor zVAD-fmk in the BV2 murine microglia cell line. More importantly, mice exposed to MPTP and further treated with Oxa12 showed protection against MPTP-induced dopaminergic neuronal loss in the SN and striatum. In conclusion, we identified Oxa12 as a hit compound that represents a new chemotype to tackle necroptosis. Oxa12 displays in vivo effects, making this compound a drug candidate for further optimization to attenuate PD pathogenesis.