Quantitative proteomic analysis reveals apoE4‐dependent phosphorylation of the actin regulating protein VASP
Background Apolipoprotein E4 (APOE4) is the major genetic risk factor for Alzheimer’s disease (AD). Current evidence suggests that neuronal expression of apoE4 is sufficient to promote neuropathology via a gain‐of‐function mechanism; however, the precise molecular mechanisms mediating this process a...
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| Veröffentlicht in: | Alzheimer's & dementia 2022-12, Vol.18 (S3), p.n/a |
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| creator | Cakir, Zeynep Polacco, Benjamin Krogan, Nevan J Mahley, Robert W Swaney, Danielle L |
| description | Background
Apolipoprotein E4 (APOE4) is the major genetic risk factor for Alzheimer’s disease (AD). Current evidence suggests that neuronal expression of apoE4 is sufficient to promote neuropathology via a gain‐of‐function mechanism; however, the precise molecular mechanisms mediating this process are still unclear. To elucidate specific protein targets and cellular pathways dysregulated due to apoE4 expression, we performed global proteomic profiling of ubiquitylation and phosphorylation signaling in Neuro‐2a cells stably expressing either apoE3 or apoE4.
Method
Neuro‐2a cells (apoE3 or apoE4) were lysed and proteins digested into peptides. Peptides were enriched for either phosphorylation or ubiquitylation post‐translational modifications, and the resulting peptides were detected and quantified using a label‐free quantitative proteomics workflow. Modification sites were detected by MaxQuant, and statistical analysis was performed using MSstats to identify significantly regulated modification sites (p |
| doi_str_mv | 10.1002/alz.061116 |
| format | Article |
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Apolipoprotein E4 (APOE4) is the major genetic risk factor for Alzheimer’s disease (AD). Current evidence suggests that neuronal expression of apoE4 is sufficient to promote neuropathology via a gain‐of‐function mechanism; however, the precise molecular mechanisms mediating this process are still unclear. To elucidate specific protein targets and cellular pathways dysregulated due to apoE4 expression, we performed global proteomic profiling of ubiquitylation and phosphorylation signaling in Neuro‐2a cells stably expressing either apoE3 or apoE4.
Method
Neuro‐2a cells (apoE3 or apoE4) were lysed and proteins digested into peptides. Peptides were enriched for either phosphorylation or ubiquitylation post‐translational modifications, and the resulting peptides were detected and quantified using a label‐free quantitative proteomics workflow. Modification sites were detected by MaxQuant, and statistical analysis was performed using MSstats to identify significantly regulated modification sites (p<0.05) between apoE3 and apoE4‐expressing cells. Immunoblotting was performed to validate protein phosphorylation sites of interest.
Result
The expression of apoE3 versus apoE4 in Neuro‐2a cells results in the differential regulation of thousands of ubiquitylation and phosphorylation sites. Gene ontology and pathway analysis of these regulated sites reveal an enrichment for a variety of cellular processes, including proteasomal biology, redox homeostasis, spindle organization, as well as cytoskeletal regulation. Accordingly, among the most differentially regulated phosphorylation sites is the vasodilator‐stimulated phosphoprotein, VASP, in which there is a 14‐fold increase in phosphorylation at position S235 in Neuro2a‐apoE4‐expressing cells (p=5.5e‐10) as compared to Neuro2a‐apoE3. This phosphorylation was reduced upon siRNA knockdown of APOE4, as well as by pharmacological inhibition of PKA, indicating that VASP S235 is phosphorylated by PKA in an apoE4‐dependent manner. VASP S235 phosphorylation results in reduced filopodia and cytoskeletal filament formation. Thus, we hypothesize that phosphorylation of VASP at S235 represents a mechanism regulating the well‐established defects in neurite outgrowth and dendritic spine formation in apoE4‐expressing neurons.
Conclusion
Our data demonstrate that VASP S235 is highly phosphorylated by PKA in apoE4‐expressing neurons compared to apoE3. Future experiments will evaluate the effect of VASP S235 phosphorylation on actin cytoskeleton remodeling and neurite outgrowth and will be extended to the evaluation of PKA inhibitors as a therapeutic opportunity in apoE4‐associated AD.</description><identifier>ISSN: 1552-5260</identifier><identifier>EISSN: 1552-5279</identifier><identifier>DOI: 10.1002/alz.061116</identifier><language>eng</language><ispartof>Alzheimer's & dementia, 2022-12, Vol.18 (S3), p.n/a</ispartof><rights>2022 the Alzheimer's Association.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Falz.061116$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Falz.061116$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,778,782,1414,27907,27908,45557,45558</link.rule.ids></links><search><creatorcontrib>Cakir, Zeynep</creatorcontrib><creatorcontrib>Polacco, Benjamin</creatorcontrib><creatorcontrib>Krogan, Nevan J</creatorcontrib><creatorcontrib>Mahley, Robert W</creatorcontrib><creatorcontrib>Swaney, Danielle L</creatorcontrib><title>Quantitative proteomic analysis reveals apoE4‐dependent phosphorylation of the actin regulating protein VASP</title><title>Alzheimer's & dementia</title><description>Background
Apolipoprotein E4 (APOE4) is the major genetic risk factor for Alzheimer’s disease (AD). Current evidence suggests that neuronal expression of apoE4 is sufficient to promote neuropathology via a gain‐of‐function mechanism; however, the precise molecular mechanisms mediating this process are still unclear. To elucidate specific protein targets and cellular pathways dysregulated due to apoE4 expression, we performed global proteomic profiling of ubiquitylation and phosphorylation signaling in Neuro‐2a cells stably expressing either apoE3 or apoE4.
Method
Neuro‐2a cells (apoE3 or apoE4) were lysed and proteins digested into peptides. Peptides were enriched for either phosphorylation or ubiquitylation post‐translational modifications, and the resulting peptides were detected and quantified using a label‐free quantitative proteomics workflow. Modification sites were detected by MaxQuant, and statistical analysis was performed using MSstats to identify significantly regulated modification sites (p<0.05) between apoE3 and apoE4‐expressing cells. Immunoblotting was performed to validate protein phosphorylation sites of interest.
Result
The expression of apoE3 versus apoE4 in Neuro‐2a cells results in the differential regulation of thousands of ubiquitylation and phosphorylation sites. Gene ontology and pathway analysis of these regulated sites reveal an enrichment for a variety of cellular processes, including proteasomal biology, redox homeostasis, spindle organization, as well as cytoskeletal regulation. Accordingly, among the most differentially regulated phosphorylation sites is the vasodilator‐stimulated phosphoprotein, VASP, in which there is a 14‐fold increase in phosphorylation at position S235 in Neuro2a‐apoE4‐expressing cells (p=5.5e‐10) as compared to Neuro2a‐apoE3. This phosphorylation was reduced upon siRNA knockdown of APOE4, as well as by pharmacological inhibition of PKA, indicating that VASP S235 is phosphorylated by PKA in an apoE4‐dependent manner. VASP S235 phosphorylation results in reduced filopodia and cytoskeletal filament formation. Thus, we hypothesize that phosphorylation of VASP at S235 represents a mechanism regulating the well‐established defects in neurite outgrowth and dendritic spine formation in apoE4‐expressing neurons.
Conclusion
Our data demonstrate that VASP S235 is highly phosphorylated by PKA in apoE4‐expressing neurons compared to apoE3. Future experiments will evaluate the effect of VASP S235 phosphorylation on actin cytoskeleton remodeling and neurite outgrowth and will be extended to the evaluation of PKA inhibitors as a therapeutic opportunity in apoE4‐associated AD.</description><issn>1552-5260</issn><issn>1552-5279</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><recordid>eNp9kM1KAzEUhYMoWKsbnyBrYTR3Mj-dZSn1Bwoq_izcDJnMTRuZJkMyrYwrH8Fn9ElMmeLSxeVcDud8i0PIObBLYCy-Es3nJcsAIDsgI0jTOErjvDj8-zN2TE68f2csYRNIR8Q8boTpdCc6vUXaOtuhXWtJhRFN77WnDrcoGk9Fa-fJz9d3jS2aGk1H25X14VzfhLI11CrarZAK2WkTasvNzjfLARqs1-nTwyk5UoGGZ3sdk5fr-fPsNlrc39zNpotIAvAsqrmUkFeqUDlWmDBeIyiMM-SyYMCRSVEkeRZXXCV1rjCtsE5BqmqiVMFjwcfkYuBKZ713qMrW6bVwfQms3C1VhqXKYakQhiH8oRvs_0mW08XbvvMLbZtv_Q</recordid><startdate>202212</startdate><enddate>202212</enddate><creator>Cakir, Zeynep</creator><creator>Polacco, Benjamin</creator><creator>Krogan, Nevan J</creator><creator>Mahley, Robert W</creator><creator>Swaney, Danielle L</creator><scope>AAYXX</scope><scope>CITATION</scope></search><sort><creationdate>202212</creationdate><title>Quantitative proteomic analysis reveals apoE4‐dependent phosphorylation of the actin regulating protein VASP</title><author>Cakir, Zeynep ; Polacco, Benjamin ; Krogan, Nevan J ; Mahley, Robert W ; Swaney, Danielle L</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c1136-d3cc17bf9f7ebe403de1fe26e3c9013e0ca94762b3f4d7fe5bed51cfb8ff932a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2022</creationdate><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Cakir, Zeynep</creatorcontrib><creatorcontrib>Polacco, Benjamin</creatorcontrib><creatorcontrib>Krogan, Nevan J</creatorcontrib><creatorcontrib>Mahley, Robert W</creatorcontrib><creatorcontrib>Swaney, Danielle L</creatorcontrib><collection>CrossRef</collection><jtitle>Alzheimer's & dementia</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Cakir, Zeynep</au><au>Polacco, Benjamin</au><au>Krogan, Nevan J</au><au>Mahley, Robert W</au><au>Swaney, Danielle L</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Quantitative proteomic analysis reveals apoE4‐dependent phosphorylation of the actin regulating protein VASP</atitle><jtitle>Alzheimer's & dementia</jtitle><date>2022-12</date><risdate>2022</risdate><volume>18</volume><issue>S3</issue><epage>n/a</epage><issn>1552-5260</issn><eissn>1552-5279</eissn><abstract>Background
Apolipoprotein E4 (APOE4) is the major genetic risk factor for Alzheimer’s disease (AD). Current evidence suggests that neuronal expression of apoE4 is sufficient to promote neuropathology via a gain‐of‐function mechanism; however, the precise molecular mechanisms mediating this process are still unclear. To elucidate specific protein targets and cellular pathways dysregulated due to apoE4 expression, we performed global proteomic profiling of ubiquitylation and phosphorylation signaling in Neuro‐2a cells stably expressing either apoE3 or apoE4.
Method
Neuro‐2a cells (apoE3 or apoE4) were lysed and proteins digested into peptides. Peptides were enriched for either phosphorylation or ubiquitylation post‐translational modifications, and the resulting peptides were detected and quantified using a label‐free quantitative proteomics workflow. Modification sites were detected by MaxQuant, and statistical analysis was performed using MSstats to identify significantly regulated modification sites (p<0.05) between apoE3 and apoE4‐expressing cells. Immunoblotting was performed to validate protein phosphorylation sites of interest.
Result
The expression of apoE3 versus apoE4 in Neuro‐2a cells results in the differential regulation of thousands of ubiquitylation and phosphorylation sites. Gene ontology and pathway analysis of these regulated sites reveal an enrichment for a variety of cellular processes, including proteasomal biology, redox homeostasis, spindle organization, as well as cytoskeletal regulation. Accordingly, among the most differentially regulated phosphorylation sites is the vasodilator‐stimulated phosphoprotein, VASP, in which there is a 14‐fold increase in phosphorylation at position S235 in Neuro2a‐apoE4‐expressing cells (p=5.5e‐10) as compared to Neuro2a‐apoE3. This phosphorylation was reduced upon siRNA knockdown of APOE4, as well as by pharmacological inhibition of PKA, indicating that VASP S235 is phosphorylated by PKA in an apoE4‐dependent manner. VASP S235 phosphorylation results in reduced filopodia and cytoskeletal filament formation. Thus, we hypothesize that phosphorylation of VASP at S235 represents a mechanism regulating the well‐established defects in neurite outgrowth and dendritic spine formation in apoE4‐expressing neurons.
Conclusion
Our data demonstrate that VASP S235 is highly phosphorylated by PKA in apoE4‐expressing neurons compared to apoE3. Future experiments will evaluate the effect of VASP S235 phosphorylation on actin cytoskeleton remodeling and neurite outgrowth and will be extended to the evaluation of PKA inhibitors as a therapeutic opportunity in apoE4‐associated AD.</abstract><doi>10.1002/alz.061116</doi><tpages>1</tpages><oa>free_for_read</oa></addata></record> |
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| title | Quantitative proteomic analysis reveals apoE4‐dependent phosphorylation of the actin regulating protein VASP |
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