Abstract
Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of cell protection genes. Classical Nrf2 activators prevent memory-loss in animal models of Alzheimer’s Disease (AD), but have side-effects in humans. Our published work identified a new class of compounds, which directly disrupt the protein-protein interaction (PPI) domain between Nrf2 and its inhibitor Keap1, as a novel and safe way for restoring Nrf2 function and preventing Aβ toxicity in mouse neurons. By developing human neuron models of Aβ toxicity, we now aim to improve clinical translation of Keap1-Nrf2 PPI disruptors for AD and to identify downstream effectors of their neuroprotective properties.
Using human induced pluripotent stem cell (hiPSC)-derived neurons, our analysis of 84 antioxidant genes (Oxidative Stress PCR Profiler Array, Qiagen), has suggested selective alterations of Nrf2 targets in response to secretomes of conditioned media from fAD-patient-derived neurons (fAD-CM) compared to healthy controls (control-CM). Only a subset of these were rescued following treatment with the Keap1-Nrf2 PPI disruptor 18e. For further functional analysis of these genes in Nrf2-mediated neuroprotection, we aimed to develop a screen-able human neuron-astrocyte model of AD. Lund Human Mesencephalic (LUHMES) cells were differentiated to neurons in 5 days and co-cultured with human astrocytes, as confirmed by immuno-fluorescence for cell type-specific markers (tubulin β-III and ALDH1L1, respectively). A preliminary study suggested that fAD-CM exerted detrimental effects on neurite outgrowth in neurons alone, and activation of Nrf2 by Keap1-Nrf2 disruptors (22h, 18e) and classical Nrf2 activator (DMF) was observed in astrocytes. Nrf2 activators were ineffective in co-cultures, however, and this appeared to be due to high basal activation of Nrf2 in astrocytes cultured in LUHMES differentiation media alone.
Future work will optimise our co-culture studies using non-contact dependent methods, for pharmacological and genetic manipulation of Nrf2 activity in astrocytes and determination of their effects on AD-related neuronal damage.
Using human induced pluripotent stem cell (hiPSC)-derived neurons, our analysis of 84 antioxidant genes (Oxidative Stress PCR Profiler Array, Qiagen), has suggested selective alterations of Nrf2 targets in response to secretomes of conditioned media from fAD-patient-derived neurons (fAD-CM) compared to healthy controls (control-CM). Only a subset of these were rescued following treatment with the Keap1-Nrf2 PPI disruptor 18e. For further functional analysis of these genes in Nrf2-mediated neuroprotection, we aimed to develop a screen-able human neuron-astrocyte model of AD. Lund Human Mesencephalic (LUHMES) cells were differentiated to neurons in 5 days and co-cultured with human astrocytes, as confirmed by immuno-fluorescence for cell type-specific markers (tubulin β-III and ALDH1L1, respectively). A preliminary study suggested that fAD-CM exerted detrimental effects on neurite outgrowth in neurons alone, and activation of Nrf2 by Keap1-Nrf2 disruptors (22h, 18e) and classical Nrf2 activator (DMF) was observed in astrocytes. Nrf2 activators were ineffective in co-cultures, however, and this appeared to be due to high basal activation of Nrf2 in astrocytes cultured in LUHMES differentiation media alone.
Future work will optimise our co-culture studies using non-contact dependent methods, for pharmacological and genetic manipulation of Nrf2 activity in astrocytes and determination of their effects on AD-related neuronal damage.
| Original language | English |
|---|---|
| Title of host publication | Glasgow Caledonian University |
| Publisher | Scottish Dementia Research Consortium |
| Publication status | Published - Sept 2020 |
