Synucleinopathies such as Parkinson's disease (PD) are hallmarked by α‐synuclein (α‐syn) pathology and neuroinflammation. This neuroinflammation involves activated microglia with increased secretion of interleukin‐1β (IL‐1β). The main driver of IL‐1β secretion from microglia is the NLRP3 inflammasome. A critical link between microglial NLRP3 inflammasome activation and the progression of both α‐syn pathology and dopaminergic neurodegeneration has been identified in various PD models in vivo. α‐Syn is known to activate the microglial NLRP3 inflammasome in murine models, but its relationship to this inflammasome in human microglia has not been established. In this study, IL‐1β secretion from primary mouse microglia induced by α‐syn fibrils was dependent on NLRP3 inflammasome assembly and caspase‐1 activity, as previously reported. We show that exposure of primary human microglia to α‐syn fibrils also resulted in significant IL‐1β secretion that was dependent on inflammasome assembly and involved the recruitment of caspase‐1 protein to inflammasome scaffolds as visualized with superresolution microscopy. While canonical IL‐1β secretion was clearly dependent on caspase‐1 enzymatic activity, this activity was less clearly involved for α‐syn‐induced IL‐1β secretion from human microglia. This work presents similarities between primary human and mouse microglia in the mechanisms of activation of the NLRP3 inflammasome by α‐syn, but also highlights evidence to suggest that there may be a difference in the requirement for caspase‐1 activity in IL‐1β output. The data represent a novel characterization of PD‐related NLRP3 inflammasome activation in primary human microglia and further implicate this mechanism in the pathology underlying PD.

Anti-apoptotic Bcl-2 family members can contribute to tumorigenesis and may convey resistance to anti-cancer regimens. Therefore, they are important targets for novel therapeutics, particularly Bcl-2 homology (BH)3 mimetics. Bcl-B (BCL-2-like protein-10) is a relatively understudied member of the Bcl-2 protein family. Its physiological function is unknown, but it has been proven to have an anti-apoptotic activity and to act as a tumor promoter in mice. In human, high Bcl-B protein expression levels correlate with poor prognosis in various carcinomas and predict treatment resistance in acute myeloid leukemia. We here report that protein expression level and anti-apoptotic activity of Bcl-B are dictated by its ubiquitination. We demonstrate that Bcl-B is polyubiquitinated at steady state, in a unique loop between the BH1 and BH2 domains. Mutagenesis identified lysine (K)128 as an acceptor site for polyubiquitin chains, and K119 and K120, but not K181, as potential ubiquitination sites. Mass spectrometry confirmed K128 as a ubiquitination site and defined the polyubiquitin chains as K48-linked, which was confirmed by linkage-specific antibodies. Accordingly, Bcl-B proved to be an instable protein that is subject to ubiquitin-dependent proteasomal degradation at steady state. At equal mRNA expression, protein expression of a lysineless, nonubiquitinated Bcl-B mutant was fivefold higher than that of wild-type Bcl-B, demonstrating that ubiquitination is a key determinant for Bcl-B protein expression levels. Ubiquitination controlled the anti-apoptotic capacity of Bcl-B, in response to a variety of conventional and novel anti-cancer drugs. Certain anti-cancer drugs, known to reduce Mcl-1 protein levels, likewise downregulated Bcl-B. Together, these data demonstrate that polyubiquitination and proteasomal turnover dictate the expression level and anti-apoptotic capacity of Bcl-B.

GH1 and GHRHR mutations are rare in Caucasian Dutch IGHD patients, which suggests the involvement of other genetic determinants in the aetiology of IGHD. IGF-I < -4.0 and peak GH levels < 5.7 mU/l are strong predictors of GH1 mutations in the studied population.