The role of protein complexes in a complex disease: molecular mechanisms of ALS

Amyotrophic Lateral Sclerosis is a devastating neurodegenerative diease caused by the selective loss of motor neurons. The pathogenic mechanism underlying the disease is largely unknown but a number of genes, proteins and cellular processes have been implicated. In this thesis we aimed to identify molecular mechanisms of ALS through genetic, proteomic and functional studies.
A number of studies have identified genetic variants causative of ALS, including mutations in SOD1, TARDBP, FUS, OPTN, UBQLN2, PFN1, C9ORF72 and TBK1. Genetic alterations in the gene PON have been suggested to be associated with ALS due to its ability to hydrolyze neurotoxic pesticides. In this thesis we show that mutations in the PON are not associated with sporadic ALS.
A neuropathologic hallmark of ALS is the presence of protein inclusions in affected motor neurons. A number of the proteins present in these aggreagtes have been identified and show a remarkable overlap with genetic findings implying; e.g. variants in the gene TARDBP segregate with the disease in a selected number of familial cases of ALS and the protein encoded by this gene, TDP-43, is present in affected motor neurons in the majority of ALS patients. Therefor, defective function of these proteins may play a role in ALS in the majority of ALS patients.
To gain insight in the proteomic changes occur in ALS, we investigated protein profiles in spinal cord material of sporadic ALS patients compared to controls. We found that ATP5D, a protein involved in mitochondrial metabolism, and Calmodulin, a protein involved in calcium homeostasis, are downregulated in ALS, implying these cellular processes in the disease.
In order to develop more efficient therapeutics in ALS, it is essential to understand how genetic alterations result in altered protein function and related disruption of cellular processes and motor neuron viability. In this thesis we investigated the interacting proteins of 6 ALS associated proteins. Our results indicate that interactomes of FUS, TDP-43 and ATXN2 on the one hand, and OPTN and UBQLN2 on the other hand, are highly overlapping and are therefor involved in common cellular processes. One of the common interactors of FUS, TDP-43 and ATXN2, FMRP, was shown to be relevant to the disease process, as overexpression of FMRP rescued FUS toxicity in a zebrafish model.
A hexanucleotide repeat expansion in an intronic region of the gene C9orf72 causes ALS, but the pathogenic mechanisms underlying this is unknown. We studied a neuron-specific C9orf72 murine knockout model and shown that downregulation of C9orf72 in neuronal cells does not lead to motor neuron loss, motor dysfunction or death, indicating that loss of C9orf72 protein is not the dominant disease mechanism in C9orf72-related ALS.