Identification of Novel Protein Targets of Dimethyl Fumarate Modification in Neurons and Astrocytes Reveals Actions Independent of Nrf2 Stabilization

Abstract

In BriefDimethyl fumarate (DMF) is a reactive fumarate ester used in the treatment of relapsing remitting multiple sclerosis; however, the neuroprotective mechanisms of DMF action are incompletely understood. The results uncover novel DMF-modified cysteine residues in neurons and astrocytes, including cytoskeletal proteins whose modulation by DMF may alter the response to neurodegenerative cues and myelination. Graphical Abstract Highlights• Dimethyl fumarate covalently modifies cysteine residues in neurons and astrocytes.• Cofilin-1, tubulin and collapsin response mediator protein 2 (CRMP2) are targets.• DMF-modified cofilin-1 reduces actin-severing ability, preserving filamentous actin.

Targeting immunometabolism as an anti-inflammatory strategy

“…39 Using mass spectrometry, Piroli et al recently identified 24 novel DMFmodified proteins in neurons and astrocytes, including cofilin-1, tubulin and collapsing response mediator protein 2 (CRMP2). 40 Using human plasmacytoid DCs, an important cell type in systemic lupus erythematosus (SLE), as well as in psoriasis and MS, Zaro et al recently described anti-inflammatory effects of DMF that are dependent on cysteine-13 of IRAK4, 41 a key enzyme in IL-1, IL-18 and Toll-like receptor (TLR) signaling. Covalent modification at this site by DMF prevents the assembly of IRAK4 and the adapter protein MyD88, leading to a reduced production of cytokines and chemokines including IFN-α and TNFα, thereby identifying cysteine 13 on IRAK4 as a vital regulatory point for adequate innate immune responses and potentially a novel therapeutic target for suppressing an overactive immune system.…”

Defective Function of α-Ketoglutarate Dehydrogenase Exacerbates Mitochondrial ATP Deficits during Complex I Deficiency

“…We then measured KGDHC activity in both the BS and OB of WT and Ndufs4 KO mice, and found that in both brain regions it was significantly lower in the KO mice (24.2 and 31.2% decrease in BS and OB, p<0.05 and 0.01 respectively, Figures 2B and 2C). Fumarate mediated reduction in the activity of the dehydrogenase was also confirmed in vitro following incubation of an isolated KGDHC standard with a range of dimethyl fumarate (DMF) concentrations, as treatment with this reactive fumarate ester also results in protein succination (Piroli et al, 2019). Figure 2D demonstrates that DMF treatment caused a dose-dependent decrease in the activity of the complex.…”

“…N1E-115 cells neuroblastoma cells (subclone N1E-115-1) were expanded in non-differentiation medium containing 90% DMEM and 10% FBS, as previously described (Piroli et al, 2016;Piroli et al, 2019). At 80% confluence, the cells were differentiated into neurons in the presence of 2% FBS and 1.25% dimethyl sulfoxide in DMEM for 5 days.…”

“…Western blotting was performed as described previously (Piroli et al, 2014;Piroli et al, 2016;Piroli et al, 2019). Ten to fifty µg of proteins were run on 12% gels (Criterion, BioRad) and transferred to PVDF membranes.…”

“…The in-gel protein digestion method used was previously described (Piroli et al, 2016;Piroli et al, 2019). Briefly, samples of the pellets obtained from the synaptosomal preparations (20 µg of protein) were resolved by SDS-PAGE in 7.5% gels; the gels were stained with Coomassie Brilliant Blue and the bands of interest in the ~48-50 kDa region were excised.…”