Mutations in LRRK2 impair NF-κB pathway in iPSC-derived neurons

Abstract

BackgroundMutations in leucine-rich repeat kinase 2 (LRRK2) contribute to both familial and idiopathic forms of Parkinson’s disease (PD). Neuroinflammation is a key event in neurodegeneration and aging, and there is mounting evidence of LRRK2 involvement in inflammatory pathways. In a previous study, we described an alteration of the inflammatory response in dermal fibroblasts from PD patients expressing the G2019S and R1441G mutations in LRRK2.MethodsTaking advantage of cellular reprogramming, we generated induced pluripotent stem cell (iPSC) lines and neurons thereafter, harboring LRRK2G2019S and LRRK2R1441G mutations. We used gene silencing and functional reporter assays to characterize the effect of the mutations. We examined the temporal profile of TNFα-induced changes in proteins of the NF-κB pathway and optimized western blot analysis to capture α-synuclein dynamics. The effects of the mutations and interventions were analyzed by two-way ANOVA tests with respect to corresponding controls.ResultsLRRK2 silencing decreased α-synuclein protein levels in mutated neurons and modified NF-κB transcriptional targets, such as PTGS2 (COX-2) and TNFAIP3 (A20). We next tested whether NF-κB and α-synuclein pathways converged and found that TNFα modulated α-synuclein levels, although we could not detect an effect of LRRK2 mutations, partly because of the individual variability. Nevertheless, we confirmed NF-κB dysregulation in mutated neurons, as shown by a protracted recovery of IκBα and a clear impairment in p65 nuclear translocation in the LRRK2 mutants.ConclusionsAltogether, our results show that LRRK2 mutations affect α-synuclein regulation and impair NF-κB canonical signaling in iPSC-derived neurons. TNFα modulated α-synuclein proteostasis but was not modified by the LRRK2 mutations in this paradigm. These results strengthen the link between LRRK2 and the innate immunity system underscoring the involvement of inflammatory pathways in the neurodegenerative process in PD.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0761-x) contains supplementary material, which is available to authorized users.

Alpha‐synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation

“…Their nature remains largely unknown but the stress that elicits the cytoprotective NF-jB AS aggregates [10] is reduced in our OLN model upon inhibition of SERCA by CPA ( Fig EV4B). NF-jB is increased in PD as determined by accumulation of p65 in midbrain dopaminergic neurons and Lewy body inclusions in PD and DLB [66][67][68][69][70] and it is also activated by disease-causing mutations in LRRK-2 when studied in iPSC-derived neurons [71]. Inhibiting NF-jB signalling in c-REL-deficient mice induces a late onset parkinsonism also comprising accumulation of aggregated AS [72] and the NF-jB inhibitor IjBia is increased early in anterior cingulate in PD [73] like we demonstrate in our cell model [10].…”

Induced pluripotent stem cell‐based modeling of mutant LRRK2‐associated Parkinson's disease

“…Significantly increased α‐syn protein levels were detected in iPSC‐derived LRRK2 G2019S mesDA‐neurons by immunoblotting (Nguyen et al., ). Subsequently, this striking finding was verified by several independent studies comparing either cultures of patient lines and controls of different genetic background (Lopez de Maturana et al., ; Nguyen & Krainc, ), or isogenic iPSC pairs (Reinhardt, Schmid et al., ).…”

“…Surprisingly, in vitro model systems have contributed to the notion that patient neurons display increased vulnerability even under normal cultivation conditions: Several studies provide evidence for increased neuronal degeneration (indicated by detection of cleaved‐caspase 3 [c‐casp3]) in cultured mesDA neurons from LRRK2 G2019S , LRRK2 I2020T , iPD background, PRKN mutant as well as SNCA triplication carriers compared to healthy controls (Lin et al., ; Ohta et al., ; Sanchez‐Danes et al., ; Suzuki et al., ). In the context of apoptosis regulation, it is important to mention that Lopez and colleagues recently described an impaired NF‐κB response in mesDA neuronal cultures of both G2019S and R1441G variants, which might point to fundamental defects in antiapoptotic signaling (Lopez de Maturana et al., ). A recent study linked LRRK2 to neuronal cell death regulation via the direct phosphorlyation and activation of apoptosis signal‐regulating kinase 1 (ASK1).…”

LRRK2 regulates innate immune responses and neuroinflammation during Mycobacterium tuberculosis infection

“…There is mounting evidence that mutations in LRRK2 contribute to immune outcomes both in the brain and in the periphery. For example, mutations in LRRK2 impair NF-κB signaling pathways in iPSCderived neurons and render rats prone to progressive neuroinflammation in response to peripheral innate immune triggers (López de Maturana et al, 2016). Additionally, chemical inhibition of LRRK2 attenuates inflammatory responses in microglia ex vivo (Moehle et al, 2012).…”