Proteomic and Metabolomic Analyses of Vanishing White Matter Mouse Astrocytes Reveal Deregulation of ER Functions

Abstract

Vanishing white matter (VWM) is a leukodystrophy with predominantly early-childhood onset. Affected children display various neurological signs, including ataxia and spasticity, and die early. VWM patients have bi-allelic mutations in any of the five genes encoding the subunits of the eukaryotic translation factor 2B (eIF2B). eIF2B regulates protein synthesis rates under basal and cellular stress conditions. The underlying molecular mechanism of how mutations in eIF2B result in VWM is unknown. Previous studies suggest that brain white matter astrocytes are primarily affected in VWM. We hypothesized that the translation rate of certain astrocytic mRNAs is affected by the mutations, resulting in astrocytic dysfunction. Here we subjected primary astrocyte cultures of wild type (wt) and VWM (2b5ho) mice to pulsed labeling proteomics based on stable isotope labeling with amino acids in cell culture (SILAC) with an L-azidohomoalanine (AHA) pulse to select newly synthesized proteins. AHA was incorporated into newly synthesized proteins in wt and 2b5ho astrocytes with similar efficiency, without affecting cell viability. We quantified proteins synthesized in astrocytes of wt and 2b5ho mice. This proteomic profiling identified a total of 80 proteins that were regulated by the eIF2B mutation. We confirmed increased expression of PROS1 in 2b5ho astrocytes and brain. A DAVID enrichment analysis showed that approximately 50% of the eIF2B-regulated proteins used the secretory pathway. A small-scale metabolic screen further highlighted a significant change in the metabolite 6-phospho-gluconate, indicative of an altered flux through the pentose phosphate pathway (PPP). Some of the proteins migrating through the secretory pathway undergo oxidative folding reactions in the endoplasmic reticulum (ER), which produces reactive oxygen species (ROS). The PPP produces NADPH to remove ROS. The proteomic and metabolomics data together suggest a deregulation of ER function in 2b5ho mouse astrocytes.

Visualization of giant virus particles using BONCAT labeling and STED microscopy

“…Because high endogenous methionine levels can impair efficient BONCAT labeling with methionine analogs such as AHA, methionine auxotrophic organisms are preferably used for this technique (Wisse et al , 2017;Zhang et al , 2017). Therefore we first examined the methionine biosynthesis coding potential in C. burkhardae using the recently published genome assembly of C. burkhardae strain RCC970-E3 (Hackl et al , 2020).…”

“…Although AHA and its alkyne analog L-homopropargylglycine (HPG) have been mostly used in methionine auxotrophic organisms to increase the efficiency of incorporation (Wisse et al , 2017;Zhang et al , 2017;Saleh et al , 2019), its use has recently been extended to the analysis of natural bacterial communities, resulting in good incorporation in active methionine prototrophic bacteria (Hatzenpichler et al , 2014;Leizeaga et al , 2017;Couradeau et al , 2019). In addition, four wild-type prototrophic eukaryotic organisms were reported to incorporate AHA or HPG: Danio rerio (Hinz et al , 2012), Caenorhabditis elegans (Ullrich et al , 2014), Arabidiopsis thaliana (Glenn et al , 2017) and the coccolithophore Emiliania huxleyi (Pasulka et al , 2018).…”

Integrated Stress Response Deregulation underlies Vanishing White Matter and is a target for therapy

“…Total RNA was extracted from mouse brain and human white matter with Trizol TM reagent (Invitrogen). Reverse transcription and qPCR were performed as described previously (71).…”

“…Membranes used for detecting phosphorylated eIF2α were blocked with 5% bovine serum and incubated with anti-Ser51-phosphorylated eIF2α antibody (Cell Signaling, D9G8). After incubating with secondary antibody (HRP-labeled anti-IgG rabbit, 1:10000, Dako, P0448), the signal was detected with an enhanced chemiluminescent substrate (SuperSignal West Femto Substrate, Fisher Scientific) and imaged with Image Studio on the LI-COR Odyssey Fc Imager (71).…”

“…Protein synthesis rates were measured in adult mouse astrocytes with AHA-incorporation and statistical analysis of the data was performed as previously described (75). Primary cultures of adult mouse astrocytes were transfected with pGL3-Gapdh and pNL1.1-Atf4 as described (75). Cells were exposed to 300 nM thapsigargin for 16 hours in absence or presence of increasing concentrations of ISRIB.…”