Aswathy Chandran-Formatted-thesis-word-TDO-corrected-FINAL corrected
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Cell type-specific effects of the transcription factors Nfe2L1 and Nrf2 in cellular models of Parkinson's disease
Parkinson’s disease is an age-related neurodegenerative disorder primarily characterized by the loss of dopaminergic neurons in the substantia nigra region of the brain and the presence of Lewy bodies, insoluble protein aggregates enriched with the presynaptic protein alpha-synuclein (aSyn). Clinically, the disease manifests as a movement disorder with varying symptoms including resting tremor, bradykinesia and postural instability. PD patients also have an array of non-motor symptoms such as swallowing and gait difficulties, REM sleep disorder, gastrointestinal distress and, in later stages, cognitive dysfunction of varying degrees. Although it has been over 200 years since PD was first described, therapies for this progressive degenerative disease are limited to symptomatic relief strategies. The identification of aSyn as a primary component of Lewy bodies and the link between SNCA (the gene encoding aSyn) and familial PD kickstarted a large body of research that established aSyn misfolding and aggregation as a key pathological factor in PD. aSyn aggregation has since been linked to dysfunction in many intraneuronal pathways including altered proteostasis and oxidative stress. Therapeutic strategies to combat this phenomenon are now an active area of investigation.
All cells contain defense mechanisms to combat various kinds of cellular stresses including proteotoxic and oxidative stress. In this thesis, I explore two such defense mechanisms regulated by the transcription factors Nfe2L1 and Nrf2 in neurons and astrocytes. Nfe2L1 and Nrf2 are two stress-induced transcription factors of the Cap’n’Collar-basic leucine zipper (CNC-bZIP) family that are responsible for regulating the expression of ubiquitin proteasome system (UPS) effectors and various antioxidant pathways. The main biological role of Nfe2L1 is considered to be the upregulation of proteasome subunit genes in response to impaired UPS function, while Nrf2 has long been established as a master regulator of oxidative stress responses. However, both Nfe2L1 and Nrf2 share a considerable degree of overlap in their transcriptomic profiles and have been shown to regulate one or both of these pathways in different cellular contexts.
In the studies summarized in Chapter 2, we explored the role of Nfe2L1 and Nrf2 overexpression on aSyn aggregation in neurons. To achieve this, we used a rat primary cortical neuron model and induced aSyn aggregation by exposing the cultures to recombinant aSyn pre-formed fibrils (PFFs), a treatment that induces the formation of mature fibrillar aggregates over time via a process of seeded self-assembly. Both Nfe2L1 and Nrf2 expression in neurons led to a decrease in aSyn aggregate burden in PFF-treated cultures and caused accelerated intraneuronal aSyn degradation. Because intracellular aSyn is known to be degraded by the proteasome, and Nfe2L1 and Nrf2 have established roles in stimulating UPS function, we hypothesized that the effect of the transcription factors on aSyn turnover could be mediated through upregulated proteasomal degradation. However, we saw no changes in the mRNA levels of proteasome subunit genes upon Nfe2L1 overexpression, nor did we observe a uniform upregulation of proteasome function across all three proteolytic activities in bulk assays involving fluorescent peptide substrates. However, Nfe2L1 expression did lead to a decrease in levels of the UPS substrate GFPu in neurons treated with the proteasome inhibitor epoxomicin, which suggests the transcription factor can have a stimulatory effect on UPS function under conditions of proteasome impairment. Further experiments aimed at monitoring transcriptomic changes and proteasome function in single cells should be carried out to confirm the mechanism by which Nfe2L1 interferes with seeded aSyn aggregation.
In the studies summarized in Chapter 3, we examined the role of Nfe2L1 and Nrf2 in mediating oxidative stress responses in neurons and astrocytes. Previous research has revealed that Nrf2 expression is restricted to astrocytes in the brain, suggesting that neuronal antioxidant responses are mediated by a different transcription factor. We assessed the ability of Nfe2L1 to perform this role in neurons by measuring changes in intraneuronal oxidative stress in response to Nfe2L1 overexpression. We found that overexpressing Nfe2L1 specifically in neurons interfered with the intraneuronal accumulation of reactive oxygen species (ROS) induced by the environmental toxin paraquat (PQ). Additionally, Nfe2L1 overexpression in astrocytes also decreased neuronal ROS levels in PQ-treated cultures in a non-cell autonomous manner that could be attributed to increased glutathione synthesis and secretion in astrocytes. Astrocytic Nrf2 expression has been shown to produce similar effects, suggesting that Nfe2L1 and Nrf2 have shared gene targets related to antioxidant defenses in astrocytes. Unlike oxidative stress responses, the proteasome subunit expression was solely regulated by Nfe2L1 in astrocytes.
Overall, the results presented in this thesis support a neuroprotective role for Nfe2L1 against PD-related pathological insults, including aSyn aggregates and the pro-oxidant paraquat. Strategies to upregulate Nfe2L1 or Nrf2 expression and transcriptional activity using gene therapy approaches or small-molecule activators could lead to promising results which target multiple pathophysiological aspects of PD. Data from orthogonal experiments such as RNA-seq and assays to measure UPS function are needed to confirm the mechanism behind these protective responses. The data presented here also suggest that Nfe2L1 and Nrf2 induce overlapping antioxidant responses in astrocytes. Additional experiments designed to knockdown expression of Nfe2L1 and Nrf2 either alone or in combination will shed a light on the extent of overlapping transcriptomic profiles of these proteins and whether their biological roles have been designed evolutionarily to exist in a coordinated fashion to protect against proteasome impairment and oxidative stress.
- Doctor of Philosophy
- Medicinal Chemistry and Molecular Pharmacology
- West Lafayette