Dissertation committee:
1. ASSOCIATE PROFESSOR PAPAZAFIRI PANAGIOTA, FACULTY OF BIOLOGY
2. RESEARCH DIRECTOR REBECCA MATSAS, HELLENIC PASTEUR INSTITUTE
3. PROFESSOR SPIROS EFTHIMIOPOULOS, FACULTY OF BIOLOGY
4. ASSOCIATE PROFESSOR DIDO VASSILACOPOULOU, FACULTY OF BIOLOGY
5. PROFESSOR LEONIDAS STEFANIS, FACULTY OF MEDICINE
6. RESEARCH DIRECTOR REGIS GRAILHE, INSTITUTE PASTEUR KOREA
7. RESEARCH DIRECTOR GEORGE PANAGIOTOU, BSRC FLEMING
Summary:
Parkinson’s disease (PD) is a complex neurodegenerative disorder affecting 2% of the world population over 65 years of age. PD is characterized by motor dysfunction related to the progressive loss of midbrain dopamine neurons while a wide range of non-motor symptoms are also present such as psychiatric manifestations and cognitive impairment. The neuropathological hallmark of PD is the presence of intracytoplasmic inclusions in neuronal cell bodies and neurites, respectively termed Lewy bodies and Lewy neurites. These are protein aggregates composed mainly of α-synuclein (αSyn), the major protein linked to sporadic PD. αSyn belongs to a class of intrinsically disordered amyloid proteins that form specific forms of oligomeric and fibrillar aggregates and exert neurotoxicity through various molecular pathways. Several point mutations (A30P, E46K, A53T, G51D) and multiplications of the SNCA locus encoding for αSyn cause autosomal dominant forms of PD. Among the different variants, the p.A53TαSyn mutation is generally considered to accelerate aggregation resulting in widespread accumulation of insoluble α-syn deposits that have been identified in the post-mortem p.A53T human brain.Despite extensive efforts in understanding PD pathogenesis, no disease modifying drugs exist. Currently only symptomatic or palliative treatments are available with none capable to prevent or slow-down disease progression. Dopamine-replacement drugs, such as levodopa, which was identified 55 years ago,are used to ameliorate motor symptoms and remain the primary and most effective treatment despite the undesired side-effects and deterioration of efficacy with disease progression. Therefore, the development of disease-modifying drugs is an urgent unmet need. Most present-day efforts in identifying novel PD therapeutics target the aggregation of misfolded αSyn as the major pathogenic factor that causes cellular toxicity. Alternative strategies to tackle early steps in neurodegeneration, particularly in an unbiased approach, have lagged behind. Recent advances in patient-derived induced pluripotent stem cell (iPSC)-based models for neurodegenerative diseases permit the detection of early, potentially triggering, pathologic phenotypes and provide amenable systems for drug discovery. In combination with high throughput high content screening technologies, these approaches open new perspectives for identification of disease-modifying compounds.
We have previously established a model of iPSC-derived neurons from patients with familial PD harboring the p.A53T αSyn mutation (G209A in the SNCA gene) that displays disease-relevant phenotypes at basal conditions. In this study, we successfully adapted this cellular system to perform the first small molecule screen on human p.A53T-neurons. Since several kinases have been implicated in PD pathology, we screened a collection of 273 small molecule kinase inhibitors to identify compounds with prospective neuroprotective properties.
We discovered that the multi-kinase inhibitor BX795 significantly reverts disease-associated phenotypes. A single treatment of patient neurons with BX795 has sustainable effects in supporting neuritic growth, restoring axonal pathology and limiting αSyn protein aggregate formation. Protection from p.A53T-associated pathology was also confirmed in human iPSC-derived neurons in which the mutation was introduced by genome editing, against isogenic wild-type controls. Strikingly, proteomics profiling by quantitative mass spectrometry revealed that BX795 treatment results in significant downregulation of a cohort of 118 proteins that are abnormally upregulated in p.A53T-neurons. To our knowledge, this study represents the first high-content drug discovery screen ans proteomics analysis performed in human p.A53T iPSC-derived neurons to identify candidate therapeutics for PD.
The p.A53T proteome examined here revealed a profound increase in proteins related to the biological processes of RNA metabolism, protein synthesis, modification and transport, protein clearance and stress response. Our proteomics analysis, identified perturbations in RNA metabolic processes that started from the nucleus and reached the ribosome. Alternative mRNA processing greatly increases the dimensions of gene expression through splicing, polyadenylation, targeted localization and post-transcriptional silencing. Neurons take advantage of all these strategies as the brain has the highest levels of alternative splicing compared to any other human tissue.This process has recently been shown to be defective in the PS19 Tau model of Alzheimer’s disease, where alternative splicing events affected genes particularly involved in synaptic transmission. Similarly, the p.A53T-proteome suggests that this process could be excessively induced in p.A53T-neurons as a number of RBPs known to be linked to αSyn aggregation have emerged, including ELAV1, ELAV3 and CELF, suggesting a possible association with the abnormal expression of synaptic genes and the defective synaptic connectivity we have previously reported in p.A53T neurons.
An excess of mRNAs coming out of the nucleus in p.A53T-neurons could explain the abnormal expression of proteins involved in translation, the next step of mRNA processing. The significant increase of components of the tRNA splicing ligase complex, various aminoacyl-tRNA synthetases, ribosomal subunits and eukaryotic translation initiation factors indicate an enhanced translation of spliced mRNAs. Moreover, in post-mortem PD brains, region and stage-dependent alterations in the machinery of protein synthesis have been reported and have been associated with α-synuclein oligomers in remaining neurons.
Dissecting further the pathways affected by BX795, we demonstrated that BX795 modulates the mTORC1 pathway to restrict excessive protein synthesis and facilitate autophagy. The mTOR kinase is a master regulator of cellular metabolism that functions in two distinct complexes: mTORC1 and mTORC2 with the first implicated in protein and lipid biosynthesis through a signaling cascade that includes SK6 and 4E-BP1 proteins. Unlike proliferating cells where this pathway is utilized for growth and division, in neurons it acts as a regulator of healthy metabolism and aging with its restriction being associated with prolonged life span and delay of age-related pathologies. p.A53T neurons have increased RPS6, IQGAP1 and RAG-GTPases, components of mTORC1 pathway and this seems to be associated with an increased translation of a subset of mRNAs that are linked to RNA metabolism and the stress response.
Concomitantly with promoting protein synthesis mTORC1 acts to repress autophagy through ULK1 phosphorylation. Autophagy has a central role in promoting health and longevity while this process is impaired in neurodegenerative diseases and αSyn pathology.The p.A53T-proteome shows that neurons are under stress as proteins involved in the UPR or the heat-shock stress response, proteasome assembly and regulation, known to be orchestrated by mTORC1 in neurons, are significantly upregulated. Restoration of numerous components of RNA metabolism and protein translation cascades by BX795 is directly related to the diminished stress response that emerges by the lower levels of UPR and heat-shock-associated proteins also conferred by this molecule. In parallel, a significant number of ubiquitin/proteasome-associated proteins were brought back to near control levels suggesting that BX795 helps misfolded protein clearance by limiting protein synthesis. This is in agreement with its demonstrated ability to decrease protein aggregates in p.A53T-neurons, as shown in this study, along with facilitation of autophagy both in SYSH-5Y cells expressing p.A53T and in patient-derived neurons.
BX795 is a multi-kinase inhibitor that targets numerous pathways, including the kinases TBK1 and PDK1. Although in our system differences in the total or phosphorylated levels of these two kinases were not observed in the presence of BX795, we cannot exclude that its effects are mediated through these two kinases as both are involved in neurodegeneration, mTOR signaling and autophagy. Interestingly, we found that BX795 could inhibit purified recombinant p70 S6K, the major kinase that phosphorylates RPS6 in vivo, indicating that the acute effect of the compound on RPS6 in p.A53T-expressing cells could be mediated through inhibition of p70 S6K. Considering that BX795 has been proposed to act through distinct mechanisms in different pathologies, future mechanistic studies should reveal its direct targets in p.A53T neurons. Nevertheless, the work presented here uniquely identifies BX795 as a promising compound that may have therapeutic potential for patients with PD and other protein conformational disorders. Further, our collective data along with previous proteomics and systems approaches shed light into the molecular and cellular pathways of αSyn proteotoxicity unveiling new disease targets for the development of combined therapeutics.
