5b34586e344c0746aef788e41ed1e04c cells-12-02800.pdf f21d5ecbbc68bfff4ec3033f4ab2d5e4abc76a68 cells-12-02800.pdf 0f9b9c7e31a614af9b3680ac6cacddfbd3222df667eb997a53a50d028845c319 cells-12-02800.pdf Title: Proteomic Analysis of Human iPSC-Derived Neural Stem Cells and Motor Neurons Identifies Proteasome Structural Alterations Subject: Background: Proteins targeted by the ubiquitin proteasome system (UPS) are identified for degradation by the proteasome, which has been implicated in the development of neurodegenerative diseases. Major histocompatibility complex (MHC) molecules present peptides broken down by the proteasome and are involved in neuronal plasticity, regulating the synapse number and axon regeneration in the central or peripheral nervous system during development and in brain diseases. The mechanisms governing these effects are mostly unknown, but evidence from different compartments of the cerebral cortex indicates the presence of immune-like MHC receptors in the central nervous system. Methods: We used human induced pluripotent stem cells (iPSCs) differentiated into neural stem cells and then into motor neurons as a developmental model to better understand the structure of the proteasome in developing motor neurons. We performed a proteomic analysis of starting human skin fibroblasts, their matching iPSCs, differentiated neural stem cells and motor neurons that highlighted significant differences in the constitutive proteasome and immunoproteasome subunits during development toward motor neurons from iPSCs. Results: The proteomic analysis showed that the catalytic proteasome subunits expressed in fibroblasts differed from those in the neural stem cells and motor neurons. Western blot analysis confirmed the proteomic data, particularly the decreased expression of the 5i (PSMB8) subunit immunoproteasome in MNs compared to HFFs and increased 5 (PSMB5) in MNs compared to HFFs. Conclusion: The constitutive proteasome subunits are upregulated in iPSCs and NSCs from HFFs. Immunoproteasome subunit 5i expression is higher in MNs than NSCs; however, overall, there is more of a constitutive proteasome structure in MNs when comparing HFFs to MNs. The proteasome composition may have implications for motor neuron development and neurodevelopmental diseases that warrant further investigation. Keywords: proteomics; induced pluripotent stem cells; differentiation; neural stem cells; motor neurons; 26S proteasome; proteasome Author: Iñaki Álvarez, Adrián Tirado-Herranz, Belén Alvarez-Palomo, Jordi Requena Osete and Michael J. Edel Creator: LaTeX with hyperref Producer: MiKTeX pdfTeX-1.40.21 CreationDate: Fri Dec 8 14:26:14 2023 CET ModDate: Fri Dec 8 14:38:13 2023 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3622763 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- BLWGYY+VnURWPalladioL Type 1 Custom yes yes no 10 0 KESBIZ+EURM10 Type 1 Builtin yes yes no 15 0 XZAZDN+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 QJTLOO+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 NTZGLL+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 AXFIVA+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 PFKOHU+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 50 0 FCFOFF+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 78 0 FCFODF+PalatinoLinotype-Roman CID TrueType Identity-H yes yes 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PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-02-08 18:18:01 CET Size: 3622763 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 407 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Human iPSC Differentiation to Motor Neurons Destination: subsection.2.1 Item: Title: Proteomics Destination: subsection.2.2 Children: Item: Title: Proteomics Sample Preparation and Measurement Destination: subsubsection.2.2.1 Item: Title: Quantitative MS Analysis Destination: subsubsection.2.2.2 Item: Title: Immunofluorescence Destination: subsection.2.3 Item: Title: Real Time PCR Destination: subsection.2.4 Item: Title: Karyotype Analysis Destination: subsection.2.5 Item: Title: Electrophysiology Destination: subsection.2.6 Item: Title: Western Blot Destination: subsection.2.7 Item: Title: Results Destination: section.3 Children: Item: Title: Human iPSC Differentiation to Motor Neurons Destination: subsection.3.1 Item: Title: Results of Proteomic Analysis of HFFs, iPSCs and NSCs Destination: subsection.3.2 Children: Item: Title: Analysis of HFFs vs. iPSCs Destination: subsubsection.3.2.1 Item: Title: Analysis of HFFs Compared to iPSCs and NSCs Destination: subsubsection.3.2.2 Item: Title: Validation of Constitutive and Immunoproteasome Findings Destination: subsection.3.3 Item: Title: Discussion Destination: section.4 Children: Item: Title: Proteomics and Western Blot Data Validation in iPSCs and MNs Destination: subsection.4.1 Item: Title: The Role of the UPS and MHC Destination: subsection.4.2 Item: Title: MHC-I Expression in Disease Destination: subsection.4.3 Item: Title: MHC Peptides Technique Destination: subsection.4.4 Item: Title: Misfolded Proteins in Neuronal Disease Destination: subsection.4.5 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.A. Info: Title: Proteomic Analysis of Human iPSC-Derived Neural Stem Cells and Motor Neurons Identifies Proteasome Structural Alterations Author: Iñaki Álvarez, Adrián Tirado-Herranz, Belén Alvarez-Palomo, Jordi Requena Osete and Michael J. Edel Subject: Background: Proteins targeted by the ubiquitin proteasome system (UPS) are identified for degradation by the proteasome, which has been implicated in the development of neurodegenerative diseases. Major histocompatibility complex (MHC) molecules present peptides broken down by the proteasome and are involved in neuronal plasticity, regulating the synapse number and axon regeneration in the central or peripheral nervous system during development and in brain diseases. The mechanisms governing these effects are mostly unknown, but evidence from different compartments of the cerebral cortex indicates the presence of immune-like MHC receptors in the central nervous system. Methods: We used human induced pluripotent stem cells (iPSCs) differentiated into neural stem cells and then into motor neurons as a developmental model to better understand the structure of the proteasome in developing motor neurons. We performed a proteomic analysis of starting human skin fibroblasts, their matching iPSCs, differentiated neural stem cells and motor neurons that highlighted significant differences in the constitutive proteasome and immunoproteasome subunits during development toward motor neurons from iPSCs. Results: The proteomic analysis showed that the catalytic proteasome subunits expressed in fibroblasts differed from those in the neural stem cells and motor neurons. Western blot analysis confirmed the proteomic data, particularly the decreased expression of the 5i (PSMB8) subunit immunoproteasome in MNs compared to HFFs and increased 5 (PSMB5) in MNs compared to HFFs. Conclusion: The constitutive proteasome subunits are upregulated in iPSCs and NSCs from HFFs. Immunoproteasome subunit 5i expression is higher in MNs than NSCs; however, overall, there is more of a constitutive proteasome structure in MNs when comparing HFFs to MNs. The proteasome composition may have implications for motor neuron development and neurodevelopmental diseases that warrant further investigation. 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