Florey Department of Neuroscience and Mental Health - Theses
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ItemAutophagy-inducing peptides as a therapy for Amyotrophic lateral sclerosis( 2022)Amyotrophic lateral sclerosis (ALS) is the most common form of motor neurone disease, where the average survival time of patients is between 2 to 5 years from symptom onset. Currently, there is no cure for ALS and the only two FDA-approved drugs, riluzole and edaravone, have a very modest effect on disease progression. Therefore, there is a great urgency for new therapeutics for this debilitating disorder. A main neuropathological hallmark of ALS is the accumulation of misfolded protein aggregates within motor neurons (MN), contributing to their degeneration. Eukaryotic cells maintain proteome homeostasis through chaperone-mediated folding, proteasomal degradation and autophagy. The only pathway that can purge the cells of these toxic protein aggregates is autophagy, an intracellular "clearance" system delivering misfolded proteins and damaged organelles to lysosomes. However, the autophagic capacity is compromised in ALS as there is an accumulation of autophagy substrates in cells. Therefore, upregulation of autophagy can have great potential as an effective treatment for ALS. This PhD study aimed to develop novel autophagy-inducing peptides that safely, efficiently and specifically increase autophagy. Firstly, a series of 22 peptides were designed based on the evolutionarily conserved domain of the Beclin-1 protein, the master regulator of autophagy. To enhance the proteolytic stability of the peptides, various modifications such as end-capping and intrachain cyclisation were introduced into the peptide sequences. Since their target is the MNs in the central nervous system, the Beclin-1-derived peptides were conjugated to a previously developed cell and blood-brain barrier (BBB)-permeable peptide (HA2-ApoE (CPP)) to enable them to cross the BBB and reach their intracellular target. The peptides were synthesised and screened in MN-like NSC-34 cells for autophagy activity. Two of the peptides, HA2-ApoE-Beclin 3 (CPP-BCN3) and HA2-ApoE-Beclin 4 (CPP-BCN4), significantly increased autophagy, even surpassing the effects of the well-accepted autophagy inducer rapamycin. Next, the two autophagy-inducing peptides were investigated for their ability to clear the protein aggregates associated with ALS in vitro. Both CPP-BCN3 and CPP-BCN4 peptides effectively reduced the levels of misfolded and aggregated mutant superoxide dismutase 1 (SOD1) protein, glycine-arginine (GR) dipeptide-repeat protein encoded by the hexanucleotide repeat expansion mutation in the C9orf72 gene, and C-terminally fragmented wild-type transactive response DNA binding protein 43 kDa (TDP-43). Consequently, peptide treatment reduced the cell death associated with mutant SOD1 expression. The most potent peptide candidate, CPP-BCN4, was next advanced in vivo for safety and autophagy-inducing efficacy. Mice were treated with CPP-BCN4 at different doses, routes of delivery and duration of treatment using an autophagy reporter mouse model. The peptide was found to be safe and well-tolerated at 10 ug/day of intracerebroventricular (ICV) infusion for 1 week and intravenous (IV) administration at 10 mg/kg for 3 times per week. The initial analysis of autophagy in the spinal MNs from this cohort of mice showed a trend towards an increase in the autophagy flux, although not statistically significant. In this ongoing study, we will assess the MN autophagy efficacy of the peptide in a larger cohort of mice. In conclusion, this study will provide the basis for a preclinical trial of autophagy peptides as a potential treatment avenue for ALS.