WPs » WP4

WP4

Workpackage 4: Proteomics

Nr

Objective

1

To optimize and validate techniques for patient iPS cell derived motor neurons, using mouse models and isolated primary motor neurons from mouse models

2

To investigate ALS aggregate formation and composition through quantitative proteomic techniques using iPS derived motor neurons

3

Search for novel proteins involved in ALS pathology by quantitative interaction analysis

4

Perform differential transcription factor binding analysis at SNPs associated with ALS

 

Large-scale proteomics studies on ALS models are relatively scarce. Thus far, investigations have mostly focused on proteins obtained from rodent tissues and cells and the reported changes in the proteome involve relatively abundant proteins. In terms of experimental design, many investigations focus on pre-symptomatic stages, aiming to discover mechanisms of disease onset rather than pathways of neurodegeneration. The effects of mSOD1 on its downstream targets can be manifested by changes in protein expression and protein-protein interactions. The changes in protein expression in NSC34G93A cells, a culture-based ALS model, were more extensive than those observed in the spinal cords of rodents with ALS-like diseases, presumably due to mitochondrial sub-proteome enrichment and differences between the systems. One of the most prominent features of proteomic changes in the spinal cord of animals affected by ALS-like diseases relates to the alteration of protein chaperones regulating protein folding and degradation pathways. In addition, proteins involved in redox regulation, cellular structure and trafficking, cell death signal transduction and oxidative response have been reported to be altered in ALS model systems.

An important breakthrough in our understanding of ALS pathogenesis was the identification of the 43 kDa TAR DNA-binding protein (TDP-43) as a major component of ubiquitinated protein aggregates found in many patients with ALS or the most common form of frontotemporal dementia called FTLD-U (frontotemporal lobar degeneration with ubiquitinated inclusions). In ALS and FTLD-U patients, TDP-43 immunoreactive inclusions are observed in the cytoplasm and nucleus of both neurons and glial cells. Mouse models with mutations in the TDP-43 gene are being generated in WP9 of this consortium, and can be used for optimization and validation of techniques that allow isolation of high quality material from tissues and iPS cells derived from ALS patients. The brains and spinal cords of patients with TDP-43 proteinopathy present a biochemical signature that is characterized by hyperphosphorylation and ubiquitination of TDP-43. With the striking exception of patients with familial ALS caused by SOD1 and FUS mutations, TDP-43 inclusions are now recognized as a common characteristic of sporadic and familial ALS patients, and provide thereby potentially an exciting link between the pathophysiology of sporadic and familial ALS. The precise roles of TDP-43 and FUS/TLS have not been fully elucidated, but both are multifunctional proteins that have been implicated in several steps of gene expression regulation including transcription, RNA splicing, RNA transport, and translation.

In genetic as well as sporadic forms of ALS, aggregates form in neural and non-neural cells. In fact, many etiologically unrelated neurodegenerative diseases are associated with the presence of abnormal protein aggregates or inclusions. Until today many possible and mutually not exclusive mechanisms have been proposed to explain the cytotoxicity of protein aggregates. These include perturbation of protein folding; dysfunction of the proteasome by undigestable, misfolded protein and interference with mitochondria and peroxisome function. However, the common features of composition and formation of aggregates in various genetic or sporadic cases of ALS remains unclear. Novel genes involved in ALS, combined with information on ALS aggregate formation, allows a thorough investigation, also from a proteomic point of view.

Studying the proteome has long been problematic, especially when compared to the wide array of possibilities for studying DNA and RNA expression, such as sequencing, oligonucleotide hybridization and amplification. Introduction of quantitative methods, like stable isotope labeling of amino acids in cell culture (SILAC; Ong et al, 2003) have allowed direct comparison of proteomes. By growing two populations of cells in culture, one with light (or normal) amino acids, and one with heavy amino acids (e.g. 13C instead of 12C), one creates a known mass shift when subsequently analyzing samples on a mass-spectrometer. The ratio of the light-heavy protein pair in mass-spectrometry data measures the relative expression of that specific protein in both samples. We have applied this technique previously to identify and quantify more than 4,000 proteins (Bonaldi et al, 2008).

Until now, proteomic approaches to investigate the proteome or the insoluble proteome fraction of wild-type or aggregate-forming ALS mutants were limited to the use of cell lines. However, since the recent technical advancement of the generation of induced pluripotent stem cells (iPS) from individual ALS patients, large-scale production of iPS derived motorneurons is possible (Dimos et al, 2009). These provide the tools for an in-depth proteomic screen as well as subproteomic analysis of disease associated aggregates using SILAC.

Summarizing, current knowledge of proteomic changes in ALS pathogenesis severely lags behind the knowledge of DNA risk factors and changes in mRNA expression. Finding new genes and developing new models calls for a reliable and quantitative way of investigating ALS proteome changes, too.

Another way to identify disease related proteins are genome wide association studies. These studies have been a powerful tool to localize genes associated with ALS. However, most associated SNPs reside in non-coding regions of the genes and affect gene transcription rather than the nature of the gene product itself. For example, one of the members of this consortium has identified six SNPs in strong linkage disequilibrium in the vicinity of the DPP6 gene (Van Es et al, 2008)

We have recently demonstrated that the SILAC technology can be exploited to study DNA-protein interactions (Mittler et al., 2009). Very recently, using this technique we have identified a previously unknown repressor protein binding to a specific DNA sequence (Butter et al, 2010). Candidates of this screen can be linked to changes in mRNA abundance between a protective/susceptible allele, thereby providing a upstream intervention point for treatment.

A further major open question in ALS has been the identification of proteins involved in disease pathogenesis. Although 9 proteins are known to be involved in ALS, genome-wide association studies have identified at least 5 further associations (reviewed by Dion et al, 2009) where the causal gene in the region has not been pinpointed due to the strong linkage disequilibrium in these genomic regions extending to dozens of candidates. The disease relevant proteins among these candidates remain to be identified.

The current proposal describes ways of how we will use several quantitative proteomic strategies to address four important areas of the ALS disease (proteome differences, aggregate formation, identification of novel pathway proteins and the effect of single nucleotide polymorphisms for genetic predisposition) within the Euro-MOTOR.

The work conducted in this work package will attempt to define proteome differences in motor neurons derived from iPS cells of ALS patients to clarify the composition of disease associated aggregates and to identify novel proteins important in ALS pathology. For all these goals, we will apply quantitative proteomic techniques developed in our laboratory.

We will assist WP8 with streamlined study on candidates to select the most promising candidates selected in WP2 and connect to WP9 with additional data for modeling.

Task 1. Proteome comparison of motor neurons generated from patient-derived iPS cells (partner 6, 11)

Within the Euro-MOTOR consortium, fibroblasts of patients carrying different mutations and genetic risk factors will soon be available. Combined with research efforts into the development of iPSC-derived MNs, we will compare the proteome changes of motor neurons from different ALS patients and unaffected controls using SILAC.

When differentiating PSCs into motor neurons, pathological changes occur including aggregate formation and increased cell death. Using differently labeled isotopes it is possible to compare cells before and after differentiation into motor neurons, and analyze pathological proteome changes that coincide with motor neuron degeneration.

These results will be integrated (WP9) with DNA, RNA and metabolic analysis (WP5, WP6) of the same cells used in other WPs.

Task 2. Unbiased proteome-wide identification of aggregate components (partner 6)

Aggregation is an important pathological hallmark of ALS disease. However, the exact contents of these aggregates is unknown. We will analyse the specific contents of iPS derived motor neuron aggregates with SILAC to improve our understanding of aggregate formation and thus disease process and progression. This will be an important basis for future ALS drug research.

Task 3. Identification of novel proteins involved in ALS by quantitative protein-protein interaction assay (partner 6)

Currently, several proteins (SOD1, ALS2, SETX, FUS, VAPB, ANG, TARDBP, DCTN1, MAPT) have been identified to be involved in ALS. We will perform a quantitative protein-protein interaction screen using these proteins as baits to identify interaction partners. This will result in candidates that can be tested in the functional models described in WP8 and can be modeled into pathways in WP9.

This screen is highly specific and can be performed for dozens of proteins. As pointed out above, genome-wide association studies identified large genomic loci which consist of dozens of genes without being able to identify individual genes due to technical limitations. We will perform at least 25 quantitative protein pull-downs which will include candidates from 2), candidates from the large scale DNA sequencing effort (WP3), pathway modelling (WP9) and identified mutations with disease phenotype (WP8)

Task 4. Establish a streamlined follow-up strategy for associated SNPs (SNP-DNA pull down) (partner 6)

WP4 will try to address the effects of SNPs on the binding of transcription factors in the relevant cellular systems, i.e. NSC34. Nuclear extracts of SILAC-labeled cells are combined with chemical synthesized DNA oligonucleotides carrying the major and minor allele of the SNP.  We have previously demonstrated that our technique can detect differential transcription factor binding at a single SNP. However, it is currently not compatible with the sample size required to identify differential transcription factor binding at dozens of SNP, the likely result of fine-mapping analysis. We will devise modifications on the current technology to allow streamlined screening. The streamlined technique will be used to perform 50 SNP pull-downs relevant to ALS (either NSC34 or patient-derived iPSCs). Target SNPs are selected in collaboration with WP 3 and screened in selected cell lines.