Targeting TFEB and TFE3 in iPSC-derived dopaminergic neurons to correct autophagic/lysosomal deficits in Parkinson's disease

Parkinson’s disease is characterised by the degeneration of dopaminergic neurons (DaNs). A key pathological hallmark within the remaining cells is the presence of proteinaceous intracellular aggregates. Clearance and recycling of damaged/aggregated proteins by the lysosome is pertinent to neuronal homeostasis and survival. Genetics, post-mortem study and disease modelling have implicated autophagic/lysosomal dysfunction as a key pathogenic mechanism contributing to PD. Mutations in PD-related genes such as GBA, LRRK2 and SNCA have all been implicated to present deficits in lysosomal catabolic pathways.
Activation of cellular catabolism by targeting master regulators of lysosomal biogenesis, TFEB and TFE3 is an attractive therapeutic strategy in PD. Although TFEB/3 activation has been demonstrated to confer neuroprotective effects, little has been done to characterise TFEB/3 function and therapeutic viability in a human, disease-relevant model of PD.
We aimed to comprehensively characterise endogenous TFEB and TFE3 in human iPSC-derived DaNs (iPSC-DaNs), before examining how genetic and pharmacological modulation can upregulate lysosomal function and rescue deficits in patient-derived iPSC-DaNs carrying GBA-N370S, LRRK2-R1441C and SNCA-Trp mutations. Strikingly, using iPSC-derived neuronal models, iPSC-derived microglia and online datasets, we found negligible TFEB expression in neuronal populations, with expression primarily associated with non-neuronal cell-types. Conversely, TFE3 was robustly expressed in neurons, making this transcription factor the prime candidate for stimulating lysosomal biogenesis in neurons to combat PD.
We developed a high-throughput qRT-PCR screen in iPSC-Ngn2 neurons to identify transcriptional regulators of TFEB in neurons. We identified histone deacetylases (HDACs) to robustly regulate neuronal TFEB expression, with HDAC inhibition stimulating TFEB expression and downstream lysosomal glucocerebrosidase function. Delineation of hit HDAC-targeting compounds, in-tandem with CRISPRi validation, indicated a redundant role between HDAC1, HDAC2 and HDAC3 to regulate TFEB expression in neurons.
Surprisingly, TFEB overexpression mainly upregulated genes associated with mitochondrial function and bioenergetics in iPSC-DaNs, demonstrating a novel, DaN- specific therapeutic function for TFEB activation in PD.
Examination of TFE3 activation in iPSC-DaNs demonstrated a major role for upregulating lysosomal biogenesis and function, more so than TFEB activation. Further, TFE3 overexpression upregulated glucocerebrosidase activity and endolysosomal function in patient-derived iPSC-DaNs that displayed perturbation in these functions. Thus, TFE3 activation is a therapeutically viable strategy for targeting lysosomal biogenesis in PD, demonstrated in a human and highly-disease relevant model.
Collectively, we demonstrate TFE3 to be the primary master regulator of lysosomal biogenesis in neurons, and activation enhanced lysosomal function in patient-derived iPSC-DaNs. We also identified HDACs as regulators of TFEB expression in neurons and present a novel, therapeutically viable approach for re-introduction of TFEB into neurons by HDAC inhibition. Our data shows TFE3 to have great therapeutic potential in PD but also provides a means to re-introduce TFEB in neurons to combat PD in the future.

Keywords: lysosome
dopaminergic neurons
TFE3
Parkinson's disease
iPSC
autophagy
TFEB
Advisor: Wade-Martins, R
Ryan, B
Hirst, W
Guardado, C