Search for: All records

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that affects neurons in the brain and spinal cord, causing loss of muscle control, and eventually leads to death. Phosphorylated transactive response DNA binding protein‐43 (TDP‐43) is the major pathological protein in both sporadic and familial ALS, forming cytoplasmic aggregates in over 95% of cases. Of the 10–15% of ALS cases that are familial, mutations in TDP‐43 represent about 5% of those with a family history. We have developed anin vitrooverexpression model by introducing three familial ALS mutations (A315T, M337V, and S379P) in the TDP‐43 (TARDBP) gene which we define as 3X‐TDP‐43. This overexpression model TDP‐43 shows deficits in autophagy flux and colocalization of TDP‐43 with stress granules. We also observe a progressive shift of TDP‐43 to the cytoplasm in this model. This overexpression model shows a reduction in solubility of phosphorylated TDP‐43 from RIPA to urea soluble. Four glycolytic enzymes, phosphoglycerate kinase one (PGK1), aldolase A (ALDOA), enolase 1 (ENO1), and pyruvate dehydrogenase kinase 1 (PDK1) show significant time‐dependent decreases in 3X‐TDP‐43 expressing cells. Shotgun proteomic analysis shows global changes in the importin subunit alpha‐1 (KPNA2), heat shock 70 kDa protein 1A (HSPA1A), and protein disulfide‐isomerase A3 (PDIA3) expression levels and coimmunoprecipitation reveals that these proteins complex with TDP‐43. Overall, these results suggest that the 3X‐TDP‐43 model may provide new insights into pathophysiology and an avenue for drug screeningin vitrofor those suffering from ALS and related TDP‐43 proteinopathies. 

Abstract Histone post‐translational modifications (PTMs) play important roles in many biological processes, including gene regulation and chromatin dynamics, and are thus of high interest across many fields of biological research. Chromatin immunoprecipitation coupled with sequencing (ChIP‐seq) is a powerful tool to profile histone PTMsin vivo. This method, however, is largely dependent on the specificity and availability of suitable commercial antibodies. While mass spectrometry (MS)–based proteomic approaches to quantitatively measure histone PTMs have been developed in mammals and several other model organisms, such methods are currently not readily available in plants. One major challenge for the implementation of such methods in plants has been the difficulty in isolating sufficient amounts of pure, high‐quality histones, a step rendered difficult by the presence of the cell wall. Here, we developed a high‐yielding histone extraction and purification method optimized forArabidopsis thalianathat can be used to obtain high‐quality histones for MS. In contrast to other methods used in plants, this approach is relatively simple, and does not require membranes or additional specialized steps, such as gel excision or chromatography, to extract highly purified histones. We also describe methods for producing MS‐ready histone peptides through chemical labeling and digestion. Finally, we describe an optimized method to quantify and analyze the resulting histone PTM data using a modified version of EpiProfile 2.0 for Arabidopsis. In all, the workflow described here can be used to measure changes to histone PTMs resulting from various treatments, stresses, and time courses, as well as in different mutant lines. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Nuclear isolation and histone acid extraction Basic Protocol 2: Peptide labeling, digestion, and desalting Basic Protocol 3: Histone HPLC‐MS/MS and data analysis