Interleukin-1β drives NEDD8 nuclear-to-cytoplasmic translocation, fostering parkin activation via NEDD8 binding to the P-ubiquitin activating site

Abstract

Background: Neuroinflammation, typified by elevated levels of interleukin-1 (IL-1) α and β, and deficits in proteostasis, characterized by accumulation of polyubiquitinated proteins and other aggregates, are associated with neurodegenerative disease independently and through interactions of the two phenomena. We investigated the influence of IL-1β on ubiquitination via its impact on activation of the E3 ligase parkin by either phosphorylated ubiquitin (P-Ub) or NEDD8.

Methods: Immunohistochemistry and Proximity Ligation Assay were used to assess colocalization of parkin with P-tau or NEDD8 in hippocampus from Alzheimer patients (AD) and controls. IL-1β effects on PINK1, P-Ub, parkin, P-parkin, and GSK3β-as well as phosphorylation of parkin by GSK3β-were assessed in cell cultures by western immunoblot, using two inhibitors and siRNA knockdown to suppress GSK3β. Computer modeling characterized the binding and the effects of P-Ub and NEDD8 on parkin. IL-1α, IL-1β, and parkin gene expression was assessed by RT-PCR in brains of 2- and 17-month-old PD-APP mice and wild-type littermates.

Results: IL-1α, IL-1β, and parkin mRNA levels were higher in PD-APP mice compared with wild-type littermates, and IL-1α-laden glia surrounded parkin- and P-tau-laden neurons in human AD. Such neurons showed a nuclear-to-cytoplasmic translocation of NEDD8 that was mimicked in IL-1β-treated primary neuronal cultures. These cultures also showed higher parkin levels and GSK3β-induced parkin phosphorylation; PINK1 levels were suppressed. In silico simulation predicted that binding of either P-Ub or NEDD8 at a singular position on parkin opens the UBL domain, exposing Ser₆₅ for parkin activation.

Conclusions: The promotion of parkin- and NEDD8-mediated ubiquitination by IL-1β is consistent with an acute neuroprotective role. However, accumulations of P-tau and P-Ub and other elements of proteostasis, such as translocated NEDD8, in AD and in response to IL-1β suggest either over-stimulation or a proteostatic failure that may result from chronic IL-1β elevation, easily envisioned considering its early induction in Down's syndrome and mild cognitive impairment. The findings further link autophagy and neuroinflammation, two important aspects of AD pathogenesis, which have previously been only loosely related.

Keywords: Alzheimer’s; Autophagy; GSK3β; IL-1β; NEDD8; Neddylation; PINK1; Parkin; Simulation interactions; Ubiquitination.

Fig. 1

Experimental evidence that regulation of parkin expression and phosphorylation is related to glial activation and excess IL-1β in Alzheimer brain and in AD Model Systems. a Parkin in neurons in brains of age-matched control patients (AMC) is diffusely distributed within the cytoplasm whereas parkin in neurons in brains of Alzheimer patients (AD) is present in rosette-like aggregates (b). c Parkin in Alzheimer brain is colocalized with tau aggregates. d Activated glia overexpressing IL-1α (brown) are immediately adjacent to parkin-immunoreactive neurons (blue), scale bar 20 μM. e Comparative levels of IL-1α, IL-1β, and parkin mRNAs in wild-type littermates or PD-APP mice. f Representative western immunoblot showing proteins from NT2 cells either treated, or not, overnight with IL-1β. g Phospho-parkin levels (P-parkin Ser₆₅) in two representative western immunoblots of proteins from NT2 cell and rat primary neuron cultures treated, or not, with IL-1β. h Quantification of parkin in untreated and treated cell cultures (n = 6), data is mean ± SEM (**p < 0.01). i, j Quantification of P-parkin levels in IL-1β treated or untreated cultures in NT2 cells (i) and primary rat neurons (j)

Fig. 2

IL-1β treatment of both NT2 cells and rat primary neurons decreased the steady-state levels of PINK1 and of Ser₆₅-phosphorylated ubiquitin (P-Ub); conversely, IL-1β increased the levels of Ser₆₅-phosphorylated parkin (P-parkin). a Representative western immunoblots depicting PINK1 levels in IL-1β treated vs untreated NT2 and rat primary neuronal cell cultures, representing 6 independent experiments. b Quantification of PINK1 levels in both NT2 cells (p = 0.01), and primary rat neurons (p = 0.05). c Representative western immunoblots of IL-1β treated vs untreated NT2 and rat primary neuronal cell cultures (n = 6 and 3 technical repeats, respectively). d Quantification of P-Ub levels in both NT2 (p = 0.03) and rat primary neurons (p = 0.005)

Fig. 3

Cytoplasmic NEDD8 levels are higher in hippocampal pyramidal neurons in Alzheimer brain than in analogous neurons in control brains, and parkin and NEDD8 are colocalized in a disease-specific manner. a Compared with their age-matched control (AMC) counterparts (n = 5), b nuclei (blue) of neurons in Alzheimer patients (n = 6) have less NEDD8 (green) (**p < 0.005) (c). d Moreover, in representative images of hippocampal pyramidal cells from these AMC and AD patients (e), colocalization of NEDD-8/Parkin (red dots depicted by PLA represent proximity of ≤ 40 nm) is less in AMC than in analogous neurons in Alzheimer patients *p < 0.05 on Wilcoxon rank sum test, n = 3 per group

Fig. 4

IL-1β treatment of primary neuronal cultures leads to NEDD8 translocation. Fluorescent immunocytochemistry of representative cultures of untreated (a) and IL-1β-treated (b) primary rat neurons (n = 6 each) depict greater translocation of NEDD8 (green) from nucleus (blue) to cytoplasm when IL-1β is present, **p < 0.01, ^††p < 0.01 (c). Quantification of western immunoblots (d) of nuclear (histone) and cytoplasmic (actin) cell fractions from untreated and IL-1β-treated cultures (n = 6 separate experiments) demonstrates the susceptibility of NEDD8 to undergo nuclear to cytoplasmic translocation in the presence of excess IL-1β, **p < 0.01, ***p < 0.01 (e). Quantification of NEDD8/parkin colocation (≤ 40 nm) in untreated NT2 cells (f) compared with IL-1β-treated NT2 cells (g) provides evidence of IL-1β-induced interaction between of parkin and NEDD8 in NT2 cells, two-tailed t test, *p < 0.05 (h). NEDD8-parkin proximity (red dots) was quantified with FIJI (ImageJ) Particle Analysis

Fig. 5

Molecular modeling indicates NEDD8 binds to parkin in the same position as P-Ub and opens up the UBL domain in a manner similar to P-Ub. a Molecular interactions for P-Ub with parkin at the P-Ub binding helix agree with the previous experimental model of P-Ub interaction with parkin [1] (b). All-atom metadynamics simulation of the P-Ub-parkin complex predicts the movement of the UBL domain exposing the Ser₆₅ from the auto-inhibited form of parkin (c–e). Protein-protein docking predicts NEDD8 interacts with the auto-inhibited form of parkin similar to that of P-Ub (f). Molecular interaction analysis indicates that NEDD8 binds to parkin at the same location as P-Ub, i.e., at the P-Ub binding helix (g). All-atom metadynamics simulation of the NEDD8-parkin complex, like that of P-Ub, also opened the UBL domain, exposing Ser₆₅ (h–j)

Fig. 6

IL-1β increases P-Parkin in a GSK3β-dependent manner. IL-1β-treated NT2 cells have increased levels of GSK3β (a, p = 4.6 × 10^− 9) and elevated GSK3β-activation, demonstrated (b) via decrease in P-GSK3β (p = 1.2 × 10^− 8) and quantified in (c). Western immunoblot analysis of NT2 cells treated with a recognized inhibitor of GSK3β attenuates IL-1β-induced parkin phosphorylation (d). Western immunoblot quantification shows a significant decrease in P-parkin levels in NT2 cells treated with GSK3β inhibitor before IL-1β treatment compared with IL-1β treatment alone (p = 0.003, one-way ANOVA, significance determined with Bonferroni-corrected α = 0.016), quantified in (e). siRNA inhibition of GSK3β in NT2 cells attenuated IL-1β-induced parkin phosphorylation (f). Quantification of P-parkin levels demonstrates that GSK3β knockdown attenuates IL-1β upregulation of parkin activation (p = 0.046, one-way ANOVA, significance determined with Bonferroni-corrected α = 0.016) quantified in (g)