Background Gelsolin is an actin‐binding protein responsible for the remodeling of the actin cytoskeleton. Gelsolin Amyloidosis, the formation of misassembled protein into insoluble amyloid fibril aggregates, is the result of point mutations that promote aberrant proteolytic cleavage. Amyloidosis can be instigated and spread by existing amyloid fibrils through the process of seeding, where existing fibrils promote the formation of new fibrils. Gelsolin fragments are prone to aggregation and systematic deposition into organs including peripheral/cranial nerves, skin, eyes, and kidneys. The D187Y mutation has been identified to promote cleavage of Gelsolin, although studies have demonstrated the mutant’s elusive aggregation propensity. Method Mutant Gelsolin peptide aggregation was monitored and characterized in vitro using various spectroscopic methods, Thioflavin T (ThT), turbidity, and Dynamic Light Scattering (DLS). Transmission electron microscopy (TEM) allowed for the identification and visualization of β‐sheet formation within aggregates Result We reported that CFILDL‐containing peptides were prone to aggregation, which may be inhibited by using small molecules. Of note is the unique behaviour of peptide mutants with respect to their aggregation propensities pointing to their biological relevance. Preliminary results suggest that seeding may induce peptide aggregation Conclusion Since other neurodegenerative proteinopathies (Alzheimer’s & Parkinson’s) exhibit similar prion‐like mechanisms of seeding that initiate aggregation, understanding the fundamentals of seeding may expose potential therapeutic targets for preventing the progression of amyloidosis. References 1) Ahmad, M., Esposto, J., Golec, C., Wu, C., & Martic‐Milne, S. (2021). Aggregation of gelsolin wild‐type and G167K/R, N184K, and D187N/Y mutant peptides and inhibition. Molecular and Cellular Biochemistry, 476, 2393–2408. https://doi.org/10.1007/s11010‐021‐04085‐6 2) Walker, L. C., Diamond, M. I., Duff, K. E., & Hyman, B. T. (2013). Mechanisms of protein seeding in neurodegenerative diseases. JAMA Neurology, 70, 304‐310. https://doi.org/10.1001/jamaneurol.2013.1453 3) Solomon, J. P., Page, L. J., Balch, W. E., & Kelly, J. W. (2012). Gelsolin amyloidosis: genetics, biochemistry, pathology and possible strategies for therapeutic intervention. Critical Reviews in Biochemistry and Molecular Biology, 47, 282–296. https://doi.org/10.3109/10409238.2012.661401 4) Ihne, S., Morbach, C., Sommer, C., Geier, A., Knop, S., & Störk, S. (2020). Amyloidosis‐the diagnosis and treatment of an underdiagnosed disease. Deutsches Ärzteblatt International, 117, 159–166. https://doi.org/10.3238/arztebl.2020.0159
Background TAR DNA‐binding protein 43 (TDP‐43) is a highly conserved nuclear protein, predominantly associated with RNA metabolism. Aberrant TDP‐43 inclusions are present in over 95% of post‐mortem brain tissue samples from Amyotrophic Lateral Sclerosis (ALS) patients. The highly disordered TDP‐43 C‐Terminal Domain (CTD) is associated with the greatest amount of disease‐related mutations and phosphorylation sites. The exact mechanism of TDP‐43 aggregation and formation into a pathological state has yet to be elucidated but is an important therapeutic target to consider. Method Full‐length phosphorylated TDP‐43 (pS410) protein was treated with 6 hours of daily agitation over 9 days in vitro. Agitation and aging took place with and without the presence of TDP‐43 RRM2‐CTD specific antibodies. Dot‐blot analysis was performed to confirm the binding of antibodies to TDP‐43. Aggregation was monitored and characterized using biophysical analysis; methods included Thioflavin T (ThT), turbidity, and transmission electron microscopy (TEM). Result Phospho‐TDP‐43 that had undergone agitation and extensive aging formed insoluble aggregates and ThT positive fibrils. The incubation with antibodies specific to the RRM2‐CTD inhibited aggregation and fibril formation in a concentration‐dependent manner. Conclusion Similar immunotherapies exist for other neurodegenerative disease‐related proteins; inhibition of TDP‐43 aggregation and fibril formation using antibodies demonstrates the potential for the epitope‐specific treatment of TDP‐43 proteinopathies. 1) Esposto, J., & Martic‐Milne, S. (2021). Biochimica et Biophysica Acta (BBA) ‐ Molecular Basis of Disease, 1867, https://doi.org/10.1016/j.bbadis.2021.166234.
Background TDP‐43 is a highly conserved protein localized to the nucleus. Under pathological conditions, it forms fibrillar aggregates, characteristic of Amyotrophic Lateral Sclerosis (ALS) progression. The exact mechanism of TDP‐43 aggregation and formation into a pathological state has yet to be elucidated, but is an important therapeutic target to consider. Methods In vitro full‐length phosphorylated TDP‐43 (pS410)protein was agitated and aged, with and without the presence of TDP‐43 specific antibodies. Immunoblotting was used to confirm the binding of antibodies to TDP‐43. Aggregation was monitored using spectroscopic methods, Thioflavin T (ThT), and turbidity. Transmission electron microscopy (TEM) visualized the formation and structure of phospho‐TDP‐43 aggregates. Results The agitation and extensive aging (days) were required to induce aggregation of insoluble phospho‐TDP‐43 aggregates and formation of ThT positive fibrils. When exposed to an antibody specific to the RRM2‐CTD domain on TDP‐43, aggregation was inhibited in a concentration‐dependent manner and fibril formation was reduced. Conclusions Inhibition of TDP‐43 aggregation and fibril formation using antibodies demonstrates the potential for epitope‐specific treatment of TDP‐43 proteinopathies. 1) Esposto, J., & Martic‐Milne, S. (2021). Phosphorylated TAR DNA‐binding protein‐43: aggregation and antibody‐based inhibition. Biochimica et Biophysica Acta (BBA) ‐ Molecular Basis of Disease, 1867, https://doi.org/10.1016/j.bbadis.2021.166234.
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