Pyroptosis-mediator GSDMD promotes Parkinson's disease pathology via microglial activation and dopaminergic neuronal death.

GSDMD-mediated pyroptosis occurs in the nigrostriatal pathway in Parkinson's disease animals, yet the role of GSDMD in neuroinflammation and death of dopaminergic neurons in Parkinson's disease remains elusive. Here, our in vivo and in vitro studies demonstrated that GSDMD, as a pyroptosis executor, contributed to glial reaction and death of dopaminergic neurons across different Parkinson's disease models. The ablation of the Gsdmd attenuated Parkinson's disease damage by reducing dopaminergic neuronal death, microglial activation, and detrimental transformation. Disulfiram, an inhibitor blocking GSDMD pore formation, efficiently curtailed pyroptosis, thereby lessening the pathology of Parkinson's disease. Additionally, a modification in GSDMD was identified in the blood of Parkinson's disease patients in contrast to healthy subjects. Therefore, the detected alteration in GSDMD within the blood of Parkinson's disease patients and the protective impact of disulfiram could be promising for the diagnostic and therapeutic approaches against Parkinson's disease.

Hypoxia inducible factor-1α regulates microglial innate immune memory and the pathology of Parkinson's disease.

Neuroinflammation is one of the core pathological features of Parkinson's disease (PD). Innate immune cells play a crucial role in the progression of PD. Microglia, the major innate immune cells in the brain, exhibit innate immune memory effects and are recognized as key regulators of neuroinflammatory responses. Persistent modifications of microglia provoked by the first stimuli are pivotal for innate immune memory, resulting in an enhanced or suppressed immune response to second stimuli, which is known as innate immune training and innate immune tolerance, respectively. In this study, LPS was used to establish in vitro and in vivo models of innate immune memory. Microglia-specific Hif-1α knockout mice were further employed to elucidate the regulatory role of HIF-1α in innate immune memory and MPTP-induced PD pathology. Our results showed that different paradigms of LPS could induce innate immune training or tolerance in the nigrostriatal pathway of mice. We found that innate immune tolerance lasting for one month protected the dopaminergic system in PD mice, whereas the effect of innate immune training was limited. Deficiency of HIF-1α in microglia impeded the formation of innate immune memory and exerted protective effects in MPTP-intoxicated mice by suppressing neuroinflammation. Therefore, HIF-1α is essential for microglial innate immune memory and can promote neuroinflammation associated with PD.

Novel Small Molecule Positive Allosteric Modulator SPAM1 Triggers the Nuclear Translocation of PAC1-R to Exert Neuroprotective Effects through Neuron-Restrictive Silencer Factor.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) exerts effective neuroprotective activity through its specific receptor, PAC1-R. We accidentally discovered that as a positive allosteric modulator (PAM) of PAC1-R, the small-molecule PAM (SPAM1) has a hydrazide-like structure, but different binding characteristics, from hydrazide for the N-terminal extracellular domain of PAC1-R (PAC1-R-EC1). SPAM1 had a significant neuroprotective effect against oxidative stress, both in a cell model treated with hydrogen peroxide (H2O2) and an aging mouse model induced by D-galactose (D-gal). SPAM1 was found to block the decrease in PACAP levels in brain tissues induced by D-gal and significantly induced the nuclear translocation of PAC1-R in PAC1R-CHO cells and mouse retinal ganglion cells. Nuclear PAC1-R was subjected to fragmentation and the nuclear 35 kDa, but not the 15 kDa fragments, of PAC1-R interacted with SP1 to upregulate the expression of Huntingtin (Htt), which then exerted a neuroprotective effect by attenuating the binding availability of the neuron-restrictive silencer factor (NRSF) to the neuron-restrictive silencer element (NRSE). This resulted in an upregulation of the expression of NRSF-related neuropeptides, including PACAP, the brain-derived neurotrophic factor (BDNF), tyrosine hydroxylase (TH), and synapsin-1 (SYN1). The novel mechanism reported in this study indicates that SPAM1 has potential use as a drug, as it exerts a neuroprotective effect by regulating NRSF.

A novel small positive allosteric modulator of neuropeptide receptor PAC1-R exerts neuroprotective effects in MPTP mouse Parkinson's disease model.

As a neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP)-preferring receptor, PAC1-R mediates effective neuroprotective activity. Based on the finding that the antibiotic doxycycline (DOX) with clinical neuroprotective activity functions as a positive allosteric modulator (PAM) of neuropeptide PACAP receptor 1 (PAC1-R), we use virtual and laboratory screening to search for novel small molecule PAMs of PAC1-R. Virtual screening is carried out using a small-molecule library TargetMol. After two-level precision screening with Glide, the top five compounds with the best predicted affinities for PAC1-R are selected and named small positive allosteric modulator 1‒5 (SPAM1‒5). Our results show that only 4-{[4-(4-Oxo-3,4-2-yl)butanamido]methyl}benzoic acid (SPAM1) has stronger neuroprotective activity than DOX in the MPP+ PD cell model and MPTP PD mouse model. SPAM1 has a higher affinity for PAC1-R than DOX, but has no antibiotic activity. Moreover, both SPAM1 and DOX block the decrease of PAC1-R level in mouse brain tissues induced by MPTP. The successful screening of SPAM1 offers a novel drug for the treatment of neurodegenerative disease targeting the PAC1-R.

Positive allosteric regulation of PAC1-R up-regulates PAC1-R and its specific ligand PACAP.

PAC1-R is a recognized preferential receptor for the neuropeptide of pituitary adenylate cyclase-activating polypeptide (PACAP), which mediates neuroprotective and nerve regenerative activities of PACAP. In this study, we found that in both PAC1R-CHO cells with high expression of PAC1R-eGFP and retinal ganglion cells (RGC-5) with the natural expression of PAC1-R, oligo-peptide PACAP(28-38) and the positively charged arginine-rich penetrating peptide TAT, as positive allosteric modulators of PAC1-R, significantly trigger the nuclear translocation of PAC1-R. The chromatin immunoprecipitation (ChIP)-PCR results show that the nuclear translocated PAC1-R binds with the promoter regions of PAC1-R and its specific ligand PACAP. The up-regulated promoter activities of PAC1-R and PACAP induced by PACAP(28-38) or TAT are positively correlative with the increase of the expression levels of PAC1-R and PACAP. Moreover, the nuclear translocation of PAC1-R induced by PACAP(28-38) or TAT is significantly inhibited by the mutation of PAC1-R on Cys25 and the palmitoylation inhibitor 2-bromopalmitate. Meanwhile, the increase in both PAC1-R and PACAP levels and the neuroprotective activities of PACAP(28-38) and TAT in MPP-induced cell model of Parkinson ' s disease are synchronously inhibited by 2-bromopalmitate, which are positively correlated with the nuclear translocation of PAC1-R induced by PACAP(28-38) or TAT. Bioinformatics analysis and motif enrichment analysis following ChIP-sequencing show that the transcription factors including SP1, Zic2, GATA1, REST and YY1 may be recruited by nuclear PAC1-R and involved in regulating the promoter activities of PAC1-R and PACAP. ChIP-sequencing and related bioinformatics analysis show that the downstream target genes regulated by the nuclear PAC1-R are mostly involved in the process of cellular stress and related to neuroprotection, neuronal genesis and development.

Blue light induces the nuclear translocation of neuropeptide receptor PAC1-R associated with the up-regulation of PAC1-R its own in reactive oxygen species associated way.

PAC1-R is neuropeptide PACAP (pituitary adenylate cyclase activating polypeptide) preferring receptor mediates the antioxidant and cytoprotective effects of PACAP. It was found in this research that in both PAC1R-CHO cells with high expression of PAC1R-eGFP and retinal ganglion cells (RGC-5) with natural expression of PAC1-R, blue light and hydrogen peroxide (H2O2) trigger the significant nuclear translocation of PAC1-R, and the nuclear translocation of PAC1-R was positive correlation with the up-regulation of expression level and promoter activity of PAC1-R its own, while red light worked much less efficiently than blue light. Reactive oxygen species (ROS) scavenger NAC (N-acetyl-L-cysteine) and palmitoylation inhibitor 2-bromopalmitate (2-BP) disturbed the nuclear shifting associated with the correlative up-regulation of PAC1 significantly, and PAC1-R mutant (M-PAC1-R) on Cys25/Ala25 displayed the significant decreased nuclear trafficking efficiency. Furthermore, the Western Blot results with the antibody raised against the C-terminal of PAC1-R showing PAC1-R in the nucleus was fragmentation hinting that C-terminal of PAC1-R with theoretical nuclear location signal (NLS) may be involved in activation of PAC1-R promoter in the nucleus. All above results indicated that PAC1-R makes the nuclear translocation to trigger the activation of PAC1-R itself promoter resulting into the up-regulation of of PAC1-R in response to the oxidative stress induced by blue light and ROS such as H2O2 .