During the initial period of the project, I have focused on the experiments described in Aim 1 and 2 and have finalized the experiments of Aim 3. Below I describe in detail work provided during this initial period in order to achieve the following deliverables.
Aim 1 : The initial results from my team demonstrate that there is a synergy between gain and loss of function of C9orf72. We have lowered the expression of C9orf72 through the usage of antisense oligonucleotides and co-expressed one of the most prevalent pathological dipeptide repeats (DPRs), GP100. Lowered expression of C9orf72 is accompanied by accumulation and aggregation of GP100 with autophagy activation by rapamycin capable of reducing DPR aggregation and motor deficits. Furthermore, we observed selective motor neuron degeneration at the level of the spinal cord upon co-expression of GP100 alongside C9orf72 reduced levels. We demonstrate by genetic experiments and proteomics results that this motor neuron death is due to mitochondrial-dependent activation of apoptosis specifically in motor neurons. This is the first vertebrate model to replicate pathological features observed in ALS patients carrying the C9orf72 repeats where both lowered C9orf72 expression and dipeptide repeat pathological features are observed. We have also developed a number of deletion mutants for C9orf72 and have identified a zebrafish homozygous mutant with adult-onset degeneration and reduced viability. Overexpression of DPRs in this mutant line leads to exacerbated toxicity and motor deficits confirming the synergy of gain and loss of function for the C9orf72 mutation.
We have developed a FUS deletion mutant line where we observe motor features associated with lowered evoked and spontaneous swimming and reduced viability at the larval stages of FUS deletion mutants where the expression of FUS has been inactivated. Importantly, in concordance with results observed in mouse model, we observed muscle defects in the FUS knockout model associated with alterations at the mitochondrial transcription with lowered rates of mitochondrial respiration measured in zebrafish homozygous mutants. Indeed a proteomics analysis reveals metabolic deficits that we are currently validating in this model as well as in pathological samples and biopsies from patients carrying FUS mutations. Similarly, we have developed zebrafish deletion mutants for the two TDP-43 orthologues in zebrafish. Unlike C9orf72 and FUS these deletion mutants do not display any major motor deficits. We are currently crossing these deletion mutants with an ALS-related mutant TDP-43 transgenic line developed by my team. Moreover, for the TDP-43 and FUS, we are currently developing kockin models that target to delete the Nuclear Localization Signal of the zebrafish FUS. Finally, we are also in the process of developing deletion line for the TBK1 gene and analyzing the phenotypic features of these mutant zebrafish.
Aim 2 : To define common pathogenic mechanisms we have assessed the autophagy response in the models described in Aim 1. Zebrafish with combined gain and loss of function of C9orf72 display altered rated of autophagic flux as measured by the LC3 GFP/RFP probe. Similarly, there is an altered upregulation of the autophagy regulator, SQSTM1/p62 with these deficits rescued upon treatment with an activator of autophagy, the mTOR inhibitor, rapamycin.
Aim 3: As mentioned above, apart rapamycin we have identified compounds that lower the neuromuscular deficits. These include the mitophagy activator, urolithin as well as the acetyl-carnitine that targets the metabolic deficits in ALS. In collaboration with clinicians, we have initiated a small clinical trial for salbutamol that targets the ER stress response.
