Targeting TFEB to correct autophagy/lysosomal deficits in Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder but disease-modifying therapies are still lacking. Genetics and pathology strongly implicate lysosomal and autophagy dysfunction in PD, making TFEB, a master-regulator of lysosomal biogenesis, an attractive target as a potential therapeutic. Importantly, activation or overexpression of TFEB prevents neurodegeneration and rescues PD related autophagy/lysosome deficits. We will assess the effect of Parkinson's disease-causing mutations on TFEB activity and regulation. Despite increasing interest in this target, the pathways involved in TFEB function in PD remain poorly understood, particularly in human neurons.
To address this, and advance TFEB as a therapeutic target, we propose three Specific Aims:
Aim 1: We will characterize TFEB localization, phosphorylation and expression levels in dopaminergic neurons (DaN) derived from induced pluripotent stem cells (iPSC) from PD patients carrying mutations in the genes LRRK2, SNCA and GBA, all readily available in Oxford. To decipher the interaction of TFEB and the lysosomal/autophagic system, we will characterize autophagic/lysosomal phenotypes present in the DaN, using live cell imaging, amongst other state of the art techniques. Moreover, we will study expression of genes regulated by TFEB as well as whole cell protein turnover by stable isotope labeling by amino acids in cell culture (SILAC) labelling and proteomics. Overexpression/activation or knockout of TFEB by siRNA or lentiviral vectors will reveal which of the differences observed between genotypes can be beneficially influenced by targeting TFEB.
Aim 2: Pathways regulating TFEB will be delineated by targeted lentiviral CRISPR-Cas9 knockout and small molecule TFEB modulators. The ability of targets within the screening library to translocate TFEB to the nucleus will be assessed by adapting an existing high-content imaging pipeline in the Wade-Martins laboratory for immortalized cell lines to DaN. The collaboration with Biogen allows the unique opportunity of designing targeted screening libraries building on hits from previously conducted high-throughput small molecule and genome wide CRISPR screens carried out at Biogen, as well as RNA sequencing data from Parkinson's iPSC-derived neurons produced by the Wade-Martins laboratory. This will identify new targets for potential gene therapy and small molecules that induce TFEB nuclear translocation.
Aim 3: We will examine the suitability of targets/molecules newly identified in Aim 2 for translation into patient care by assessing their ability to reverse TFEB-susceptible phenotypes identified in Aim 1. Since alpha-synuclein pathology is a defining phenotype of PD across genotypes, we will subsequently intend to reduce alpha -synuclein fibril toxicity in PD DaN with the most successful compounds/targets. The best compounds/targets will be assayed for in vivo PK/PD and efficacy verified in a pre-formed fibril injection humanized alpha-synuclein overexpressing mouse model previously established in Oxford.
The proposed collaboration will provide data in complex human neuronal and humanized rodent models to support Biogen and Oxford together advancing TFEB as a therapeutic target. The work is highly aligned with MRC strategic priorities by benefiting the public by identifying new drug targets and also serves the wider scientific community by increasing our understanding of the molecular pathways involved in regulating this important transcription factor.