Plasma-borne indicators of inflammasome activity in Parkinson’s disease patients

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by motor and non-motor symptoms and loss of dopaminergic neurons of the substantia nigra. Inflammation and cell death are recognized aspects of PD suggesting that strategies to monitor and modify these processes may improve the management of the disease. Inflammasomes are pro-inflammatory intracellular pattern recognition complexes that couple these processes. The NLRP3 inflammasome responds to sterile triggers to initiate pro-inflammatory processes characterized by maturation of inflammatory cytokines, cytoplasmic membrane pore formation, vesicular shedding, and if unresolved, pyroptotic cell death. Histologic analysis of tissues from PD patients and individuals with nigral cell loss but no diagnosis of PD identified elevated expression of inflammasome-related proteins and activation-related “speck” formation in degenerating mesencephalic tissues compared with controls. Based on previous reports of circulating inflammasome proteins in patients suffering from heritable syndromes caused by hyper-activation of the NLRP3 inflammasome, we evaluated PD patient plasma for evidence of inflammasome activity. Multiple circulating inflammasome proteins were detected almost exclusively in extracellular vesicles indicative of ongoing inflammasome activation and pyroptosis. Analysis of plasma obtained from a multi-center cohort identified elevated plasma-borne NLRP3 associated with PD status. Our findings are consistent with others indicating inflammasome activity in neurodegenerative disorders. Findings suggest mesencephalic inflammasome protein expression as a histopathologic marker of early-stage nigral degeneration and suggest plasma-borne inflammasome-related proteins as a potentially useful class of biomarkers for patient stratification and the detection and monitoring of inflammation in PD

Molecular Mechanisms of Notch Signaling Governing Vascular Smooth Muscle Cell Proliferation

Vascular smooth muscle cell (VSMC) proliferation is an important feature of atherosclerotic plaque formation and restenosisl. Notchl, Notch2, Notch3 and Notch4 are up-regulated in adult intimal smooth muscle cells and regenerating endothelial cells following injury using the rat balloon catheter denudation model2. In this study, the objective was to determine the mechanisms by which Notch signaling regulates the proliferation of VSMC. We employed an in vitro VSMC model for Notch gain-offunction experiments. We used this model to assess alterations in VSMC proliferation using growth curve and DNA synthesis analysis following expression of the intracellular -do mains (m)o f Notchl (NlICD) and Notch4 (N4ICD) which act as constitutively active signaling receptors, or expression of the downstream effector HES-related transcript-2 ( H R T ~ )O~v.e r-expression of NlICD, N4ICD and HRT2 was demonstrated to positively regulate S-phase entry in a rat smooth muscle cell line (PAC-1). Expression of NlICD and N4ICD was analyzed by quantitative PCR and shown to up-regulate the VSMC specific Notch target genes HRTl and HRT2. The loss of Go/Glarrest was associated with a reduction in protein levels of the cyclin dependent kinase inhibitors p27 and p21. Over-expression of p27 resulted in a dose dependent rescue of the Notchlinduced loss of cell cycle regulation. Finally, direct repression of the p27 promoter by HRT2 was demonstrated using reporter assays, gel retardation assays and chromatin immunoprecipitation. Transcriptional repression is demonstrated to occur within the minimal p27 promoter region 435 - 774bp upstream of the start codon, and mutational analysis demonstrates that repression is dependent on a conserved class-C domain within the minimal promoter region. In order to assess the relevance of in vitro Notch induced proliferation to other smooth muscle hyperplasias, human smooth muscle neoplasms were examined in order to determine whether Notch receptorslligands were expressed. Using in situ hybridization, expression of Notch receptors, particularly Notch 1 and 2 as well as the Notch ligand Jagged 1 was observed in both gastrointestinal stromal tumors and leiomyosarcomas. Lastly, in order to identify novel targets of Notch signaling in smooth muscle cells microarray analysis of human aortic smooth muscle cells was performed following transduction with HRTl and HRT2 adenovirus. Microarray experiments verified the repression of p27 by HRTl and HRT2 in primary human cells. In addition, several novel targets of HRT transcription factors were identified including Jaggedl, Sprouty2 and Sprouty4, which will be useful for generating hypotheses designed to identify smooth muscle-specific Notch signaling pathways

Behavioral abnormalities and Parkinson’s-like histological changes resulting from Id2 inactivation in mice

SUMMARY Characterizing dopaminergic neuronal development and function in novel genetic animal models might uncover strategies for researchers to develop disease-modifying treatments for neurologic disorders. Id2 is a transcription factor expressed in the developing central nervous system. Id2−/− mice have fewer dopaminergic neurons in the olfactory bulb and reduced olfactory discrimination, a pre-clinical marker of Parkinson’s disease. Here, we summarize behavioral, histological and in vitro molecular biological analyses to determine whether midbrain dopaminergic neurons are affected by Id2 loss. Id2−/− mice were hyperactive at 1 and 3 months of age, but by 6 months showed reduced activity. Id2−/− mice showed age-dependent histological alterations in dopaminergic neurons of the substantia nigra pars compacta (SNpC) associated with changes in locomotor activity. Reduced dopamine transporter (DAT) expression was observed at early ages in Id2−/− mice and DAT expression was dependent on Id2 expression in an in vitro dopaminergic differentiation model. Evidence of neurodegeneration, including activated caspase-3 and glial infiltration, were noted in the SNpC of older Id2−/− mice. These findings document a novel role for Id2 in the maintenance of midbrain dopamine neurons. The Id2−/− mouse should provide unique opportunities to study the progression of neurodegenerative disorders involving the dopamine system

Integration of Notch 1 and calcineurin/NFAT signaling pathways in keratinocyte growth and differentiation control.

The Notch and Calcineurin/NFAT pathways have both been implicated in control of keratinocyte differentiation. Induction of the p21(WAF1/Cip1) gene by Notch 1 activation in differentiating keratinocytes is associated with direct targeting of the RBP-Jkappa protein to the p21 promoter. We show here that Notch 1 activation functions also through a second Calcineurin-dependent mechanism acting on the p21 TATA box-proximal region. Increased Calcineurin/NFAT activity by Notch signaling involves downregulation of Calcipressin, an endogenous Calcineurin inhibitor, through a HES-1-dependent mechanism. Besides control of the p21 gene, Calcineurin contributes significantly to the transcriptional response of keratinocytes to Notch 1 activation, both in vitro and in vivo. In fact, deletion of the Calcineurin B1 gene in the skin results in a cyclic alopecia phenotype, associated with altered expression of Notch-responsive genes involved in hair follicle structure and/or adhesion to the surrounding mesenchyme. Thus, an important interconnection exists between Notch 1 and Calcineurin-NFAT pathways in keratinocyte growth/differentiation control