Proteomic and metabolomic analyses of vanishing white matter mouse astrocytes reveal deregulation of ER functions

Lisanne E. Wisse, Renske Penning, Esther A. Zaal, Carola G.M. van Berkel, Timo J. ter Braak, Emiel Polder, Justin W. Kenney, Christopher G. Proud, Celia R. Berkers, Maarten A.F. Altelaar, Dave Speijer, Marjo S. van der Knaap, Truus E.M. Abbink

Research output: Contribution to journalArticleAcademicpeer-review

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.

Original languageEnglish
Article number411
JournalFrontiers in Cellular Neuroscience
Volume11
DOIs
Publication statusPublished - 20 Dec 2017

Cite this

Wisse, Lisanne E. ; Penning, Renske ; Zaal, Esther A. ; van Berkel, Carola G.M. ; ter Braak, Timo J. ; Polder, Emiel ; Kenney, Justin W. ; Proud, Christopher G. ; Berkers, Celia R. ; Altelaar, Maarten A.F. ; Speijer, Dave ; van der Knaap, Marjo S. ; Abbink, Truus E.M. / Proteomic and metabolomic analyses of vanishing white matter mouse astrocytes reveal deregulation of ER functions. In: Frontiers in Cellular Neuroscience. 2017 ; Vol. 11.
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title = "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.",
keywords = "AHA, Astrocytes, eIF2B, Endoplasmic reticulum, Metabolomics, SILAC, Vanishing white matter",
author = "Wisse, {Lisanne E.} and Renske Penning and Zaal, {Esther A.} and {van Berkel}, {Carola G.M.} and {ter Braak}, {Timo J.} and Emiel Polder and Kenney, {Justin W.} and Proud, {Christopher G.} and Berkers, {Celia R.} and Altelaar, {Maarten A.F.} and Dave Speijer and {van der Knaap}, {Marjo S.} and Abbink, {Truus E.M.}",
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Proteomic and metabolomic analyses of vanishing white matter mouse astrocytes reveal deregulation of ER functions. / Wisse, Lisanne E.; Penning, Renske; Zaal, Esther A.; van Berkel, Carola G.M.; ter Braak, Timo J.; Polder, Emiel; Kenney, Justin W.; Proud, Christopher G.; Berkers, Celia R.; Altelaar, Maarten A.F.; Speijer, Dave; van der Knaap, Marjo S.; Abbink, Truus E.M.

In: Frontiers in Cellular Neuroscience, Vol. 11, 411, 20.12.2017.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Proteomic and metabolomic analyses of vanishing white matter mouse astrocytes reveal deregulation of ER functions

AU - Wisse, Lisanne E.

AU - Penning, Renske

AU - Zaal, Esther A.

AU - van Berkel, Carola G.M.

AU - ter Braak, Timo J.

AU - Polder, Emiel

AU - Kenney, Justin W.

AU - Proud, Christopher G.

AU - Berkers, Celia R.

AU - Altelaar, Maarten A.F.

AU - Speijer, Dave

AU - van der Knaap, Marjo S.

AU - Abbink, Truus E.M.

PY - 2017/12/20

Y1 - 2017/12/20

N2 - 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.

AB - 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.

KW - AHA

KW - Astrocytes

KW - eIF2B

KW - Endoplasmic reticulum

KW - Metabolomics

KW - SILAC

KW - Vanishing white matter

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U2 - 10.3389/fncel.2017.00411

DO - 10.3389/fncel.2017.00411

M3 - Article

VL - 11

JO - Frontiers in Cellular Neuroscience

JF - Frontiers in Cellular Neuroscience

SN - 1662-5102

M1 - 411

ER -