(d) Quantitation of cleaved PARP protein level from primary neurons-astrocyte coculture cell extracts. showing the quantitation of NOD2 protein level normalized to actin in the presence of increasing dosage of parkin-Myc plasmid described in Fig 4b. (b) HEK293T cells were transfected with expression constructs encoding Flag-tagged parkin and HA-tagged NOD2 and incubated with or without the proteasome inhibitor MG132. The immunoprecipitation was performed using an anti-Flag antibody and anti-Flag and anti-Myc antibodies were employed for subsequent immunoblot analysis of the NOD2 and parkin, respectively. Representative immunoblot showing evidence of lower electrophoretic mobility NOD2-Flag protein bands (depicted with the vertical line) when NOD2 was coexpressed with parkin in the presence of the proteasome inhibitor MG132. (c) Quantitation of ubiquitinated NOD2 in the coimmunoprecipitation experiment described in Fig. 4c with indicated plasmids in the presence or absence of MG132. Statistical difference was assessed by students t-test. *p 0.05, compared to the corresponding control. All experiments were repeated 3 times. IP: immunoprecipitation. WB: western blot. NIHMS974194-supplement-Supp_FigS4.tif (4.4M) GUID:?441EB6E2-673B-45AB-BC9D-0F7C6AC1CAB7 Supp figS2: Figure S2. (a) Quantitation of LDH released from SHSY5Y cells that were transduced with control or parkin shRNA lentivirus after exposure to BDNF and thapsigargin (ER stress). Statistical difference was assessed by students t-test. *p 0.05, compared to the control. All experiments were repeated 3 times. NIHMS974194-supplement-Supp_figS2.tif (488K) GUID:?7F3D2615-7CB9-4895-90F8-CECA07C94B72 Supp legends. NIHMS974194-supplement-Supp_legends.docx (16K) GUID:?2A27ED6B-0D70-4622-B6E1-D6A4C71250CB Abstract Loss of substantia nigra dopaminergic neurons results in Parkinson disease (PD). Degenerative PD usually presents in the seventh decade whereas genetic disorders, including mutations in predispose to early-onset PD. encodes the parkin E3 ubiquitin ligase which confers pleotropic effects on mitochondrial and cellular fidelity and as a mediator of endoplasmic reticulum (ER) stress signaling. Although the majority of studies investigating ameliorative effects of parkin focus on dopaminergic neurons we found that astrocytes are enriched with parkin. Furthermore, astrocytes deficient in parkin display stress-induced elevation of nucleotide-oligomerization Ureidopropionic acid domain receptor 2 (NOD2), a cytosolic receptor integrating ER stress and inflammation. Given the neurotropic and immunomodulatory role of astrocytes we reasoned that parkin may regulate astrocyte ER stress and inflammation to control neuronal homeostasis. We show that, in response to ER stress, parkin knockdown astrocytes exhibit exaggerated ER stress, JNK activation and cytokine release, and reduced neurotropic factor expression. In coculture studied we demonstrate that dopaminergic SHSY5Y cells and primary neurons with the presence of parkin depleted astrocytes are more susceptible to ER stress and inflammation-induced apoptosis than wildtype astrocytes. Parkin interacted with, ubiquitylated and diminished Ureidopropionic acid NOD2 levels. Additionally, the genetic induction of parkin ameliorated inflammation in NOD2 expressing cells and knockdown of NOD2 in astrocytes suppressed inflammatory defects in parkin deficient astrocytes and concurrently blunted neuronal apoptosis. Collectively these data identify a role for parkin in modulating NOD2 as a regulatory node in Ureidopropionic acid astrocytic control of neuronal homeostasis. value 0.05 was considered statistically significant. 3 RESULTS 3. 1 Astrocyte restricted depletion of parkin augments neuronal ER stress and inflammation-induce injury To assess the role of parkin in astrocytic neurotropic function, primary astrocytes were cultured from parkin WT and KO mice brains. The absence of parkin expression in KO astrocytes was confirmed by quantitative RT-PCR and immunoblot analysis (Supporting Information, Figure S1a,b). To test if parkin loss impacts astrocyte neurotropic function, primary astrocyte and SHSY5Y cocultures were established in transwells and cell death was monitored by measuring lactate dehydrogenase (LDH) secreted into the coculture mass media. In the lack of stressors, coculturing dopaminergic SHSY5Y cells with either WT or parkin KO astrocytes didn’t impact cell TXNIP success (Amount 1a). Additionally, contact with dopaminergic neurotoxins including 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) under these circumstances resulted in very similar degrees of cell death.