Effects of overexpression of the ALS associated protein TDP-43 and its mutant on the axonal and somal proteome in compartimentalized primary cortical neurons of rats
by Leonie Sophie Angerer
Date of Examination:2024-08-23
Date of issue:2024-08-22
Advisor:PD Dr. Jan C. Koch
Referee:PD Dr. Jan C. Koch
Referee:Prof. Dr. Ralph Kehlenbach
Persistent Address:
http://dx.doi.org/10.53846/goediss-10665
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Abstract
English
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of the motor nervous system. During its course, the function of the upper and lower motor neurons is predominantly lost. It is a very severe disease with a life-time risk of 2.6 in 1000 and a rapid disease progression, that results in death in median within four years. Symptoms of the disease include a diverse picture such muscle weakness, atrophy, fasciculation, hyperreflexia, muscle spasms and more. Most ALS cases are of sporadic nature, however about 10% of the cases can be traced back to distinct gene mutations e.g., in the TAR DNA binding protein (TDP-43). No distinct pathological mechanism has been found thus far, that explains the very diverse picture of ALS. However, many pathological mechanisms are in question. Because of the uncertainty regarding ALSs origin, therapeutic options are very limited, with only a few symptomatic therapies available. TDP-43 is a heterogenous nuclear ribonucleoprotein, that is very diverse in its functions. In health, it is predominantly located in the nucleus and supports the cell in various ways. It is essential for RNA related pathways such as transcription, translation, transportation, and splicing. Furthermore, it takes part in nucleocytoplasmic shuttling, the formation of stress granules and transportation of this granules. In ALS, TDP-43 is shifted towards the cytosol and forms protein inclusion bodies, that can be found in 97% of all ALS cases including sporadic and familial ALS. Other pathological TDP-43 related pathways include a disrupted nucleocytoplasmic shuttling, RNA alterations, mitochondrial dysfunctions, and degeneration of axons. The latter is highly relevant because axonal degeneration is an early indicator of the disease. TDP-43 can be dysregulated in its wildtype form and in mutations such as M337V, that leads to an even higher number of aggregates in the cytoplasm and increased velocity. This study aims to give a comprehensive analysis of the somal and axonal proteome in wildtype and mutant TDP-43 overexpressing cortical neurons of embryonic rats in comparison to a control. The results show changes and similarities between the wildtype and mutant TDP-43 and differences between the axonal and the somal compartment. They confirm that there are in fact differences between wildtype and mutant TDP-43 overexpression, especially in the axonal compartment and show effects that the overexpression of TDP-43 has on different aspects of cellular homeostasis. For the proteomic analysis, cortical neurons of embryonic rats were cultivated in microfluidic chambers, that made a distinct separation of the proteome of cell bodies and the axons possible. Afterwards they were transduced with viral adeno-associated vectors that overexpressed wildtype and mutant TDP-43 and only EGFP as control. TDP-43 overexpression has been confirmed through immunocytochemistry, Western Blot and proteomic data. Bioinformatical analysis has been performed using the bioinformatical software DAVID, KEGG and STRING, that make visualization of the highly complex datasets possible. In the comparison of the somal and axonal proteome for wildtype and mutant TDP-43 overexpressing cells, mostly proteins were altered regarding lysosome and synapse related pathways. Comparing wildtype and mutant in the somal compartment revealed most statistical relevant dysregulated proteins behave in a similar way, therefore being equally higher or lower abundant. The higher abundant proteins in the somal compartment for wildtype and mutant were predominantly connectable to Isopeptide bonds, messenger RNA metabolism and fatty acid metabolism. Especially mRNA metabolism and fatty acid metabolism are subjects closely linked to ALS pathology. The lower abundant proteins in the somal compartment in wildtype and mutant were linked to synaptic alterations. Comparing wildtype and mutant in the axonal compartment highlighted the differences between those two TDP-43 altering groups. Here, the majority of significantly altered proteins were higher abundant in the wildtype and lower abundant in the mutant. They were mostly related to axon guidance, synapse and microtubules. Similar behaving higher abundant proteins for wildtype and mutant in the axon were connected to isopeptide bond and endocytosis. Lower abundant proteins in the axon regarding wildtype and mutant pointed to the respiratory chain in the mitochondria, where proteins related multiple complexes were lower abundant. Throughout all analyzed groups hints towards cytoskeletal impairment were found, especially regarding actin, intermediate filaments, and microtubules. This study confirms previous findings of several different aspects of TDP-43 pathology and ALS. However, it also identifies more distinct research targets like the fatty acid metabolism and SUMOylation.
Keywords: ALS; TDP-43; Amyotrophic lateral sclerosis; Proteomics
