Biology subjects

Ayoubi, R.

Publications and source records attributed to Ayoubi, R..

4 recordsLinked to original sources

bioRxiv2025

Cell type specific analysis of ALS associated proteins reveals immune-regulated targets

A substantial fraction of amyotrophic lateral sclerosis (ALS)-associated proteins remain poorly characterized, in part because of the limited availability of validated research antibodies. We established knockout (KO)-based antibody characterization workflows and demonstrated that widely used antibodies against the major ALS-associated protein C9orf72 lacked specificity (Laflamme et al., 2019). We subsequently scaled this framework to systematically benchmark research antibodies, revealing that up to 61% fail to perform as recommended by manufacturers (Ayoubi et al., 2023). Here, we extend this approach by establishing the ALS-Reproducible Antibody Platform (ALS-RAP), a comprehensive effort to generate a publicly available dataset of KO-validated antibodies targeting proteins encoded by ALS risk genes. In total, we characterized 303 antibodies against 33 ALS-associated proteins to identify high-quality reagents for use in western blot, immunoprecipitation, and immunofluorescence. Using these antibodies, we profiled protein levels across human induced pluripotent stem cell (iPSC)-derived and primary neurological cell types. These analyses revealed diverse cellular distributions and higher levels of several ALS-associated proteins in glial populations, consistent with emerging evidence for immune contributions to ALS. Together, ALS-RAP provides a validated antibody toolbox and protein expression resource to support the study of ALS-associated proteins.

neuroscience↗

bioRxiv2023

Assessing the performance of commercial reagent antibodies

Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, demonstrates that: i) more than 50% of all antibodies failed in one or more tests, ii) yet, [~]50-75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first such study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.

molecular biology↗

bioRxiv2023

A dual hit of α-synuclein internalization and immune challenge leads to formation and maintenance of Lewy body-like inclusions in human dopaminergic neurons

Lewy bodies (LBs), rich in -synuclein, are a hallmark of Parkinsons disease (PD). Understanding their biogenesis is likely to provide insight into the pathophysiology of PD, yet a cellular model for LB formation remains elusive. The realization that the immune challenge is a trigger for neurodegenerative diseases has been a breakthrough in the understanding of PD. Here, iPSC-derived human dopaminergic (DA) neurons from multiple healthy donors were found to form LB-like inclusions following treatment with - synuclein preformed fibrils, but only when coupled to an immune challenge (interferon-gamma or interleukin-1 beta) or when co-cultured with activated microglia. Human cortical neurons derived from the same iPSC lines did not form LB-like inclusions. Exposure to interferon-gamma impairs autophagy in a lysosomal-specific manner in vitro, similar to the disruption of proteostasis pathways that contribute to PD. We find that lysosomal membrane proteins LAMP1 and LAMP2 and transcription factors regulating lysosomal biogenesis and function are downregulated in DA but not cortical neurons. Finally, due to the excellent sample preservation afforded by cells compared to post-mortem PD brain tissue, we conclude that the LB-like inclusions in DA neurons are membrane-bound, suggesting they are not limited to the cytoplasmic compartment. In Brief O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/542776v3_figabs.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1f41855org.highwire.dtl.DTLVardef@69798dorg.highwire.dtl.DTLVardef@ab55bdorg.highwire.dtl.DTLVardef@8a16b8_HPS_FORMAT_FIGEXP M_FIG C_FIG Bayati et al. identify that iPSC-derived dopaminergic neurons undergoing a dual hit treatment of exogenous -synuclein fibrils and proinflammatory cytokines form Lewy body-like inclusions. The dual hit treatment also led to the downregulation of lysosomal proteins. Characterization of inclusions revealed that inclusions were membrane-bound and LC3B-positive, suggesting they are dysfunctional autophagosomes. HighlightsO_LI-synuclein preformed fibril administration coupled with Interferon-gamma exposure leads dopaminergic neurons to form Lewy body-like inclusions C_LIO_LIInclusions are filamentous, membranous, and filled with aberrant organelles C_LIO_LIImpaired autophagic flux and downregulation of TFEB, NRF2, LAMP1, and LAMP2 correlated with inclusion formation C_LIO_LIActivation of NRF2 through the treatment of neurons with the antioxidant perillaldehyde, prevents inclusion formation C_LI

neuroscience↗

bioRxiv2021

Role of the ubiquitin ligase ITCH in clathrin-mediated endocytosis of the epidermal growth factor receptor

Once activated by ligand, epidermal growth factor receptor (EGFR) is endocytosed in clathrin-coated pits. ITCH is an E3 ubiquitin ligase that interacts with and ubiquitinates several proteins involved in clathrin-mediated endocytosis (CME) including endophilin. To further investigate the function of ITCH in EGFR endocytosis, the internalization of fluorescent EGF was measured in ITCH-/- HeLa cells. In the absence of ITCH, there was a significant decrease in the CME of EGF. Rescue experiments using wild-type ITCH confirmed the importance of the protein for normal EGF uptake. ITCH point mutations that disrupt the interaction of ITCH with endophilin failed to rescue the defects in EGFR uptake, as did a non-catalytic form of ITCH. ITCH-/- cells also displayed a delay in the rate of phospho-EGFR degradation as well as prolonged ERK1/2 signaling. Our study uncovers a pathway regulating EGFR trafficking and reveals for the first time that the protein ITCH is required for CME of EGFR.

cell biology↗