Jennifer Hensel Thesis
Reason: Preparing multiple aspects for publication
until file(s) become available
Role of alpha-synuclein in CNS diseases: pre-clinical modeling and biomarker analysis
Parkinson’s disease (PD) is an age-related neurodegenerative disorder characterized by pathological features that include the selective loss of dopaminergic (DA) neurons in the substantia nigra (SN) region of the midbrain and the presence of intraneuronal Lewy body and Lewy neurite inclusions primarily comprised of fibrillar forms of the pre-synaptic protein alpha-synuclein (aSyn). aSyn aggregation has been implicated as a critical event in PD pathogenesis, and mutant forms of aSyn are associated with familial, early-onset forms of the disease. PD presents clinically as a movement disorder through appearance of its cardinal motor symptoms of bradykinesia, rigidity, postural instability, and resting tremor. However, upon manifestation of these clinical symptoms, over 50% of the DA nigral neurons have been lost, suggesting that PD neuropathology likely begins 10-15 years prior to the clinical onset. Current PD treatment is focused on symptomatic therapy through dopamine replacement strategies, and there are currently no medications available to prevent or slow disease progression.
A major hurdle to developing disease-modifying therapies for PD is a lack of knowledge of the molecular phenomena responsible for the death of DA neurons in the SN. Multiple cellular pathways that are dysregulated in PD include protein clearance systems and oxidative stress responses. Changes in these systems by environmental or genetic perturbations cause increased aSyn aggregation, which in turn leads to increased oxidative and proteasomal stress, thereby generating a vicious cycle culminating in the death of nigral DA neurons. Therefore, strategies to activate these protective pathways have the potential to reduce aSyn aggregation and halt neurodegeneration. One such mechanism is through the activation of the stress-induced transcription factor, Nfe2L1. Nfe2L1 is a cap ‘n’ collar basic leucine zipper (CNC-bZIP) transcription factor that forms heterodimers with small Maf proteins. In turn, the heterodimers bind to antioxidant response element (ARE) sequences in the promoter regions of cytoprotective genes, such as genes encoding proteasome subunits and proteins involved in the glutathione synthesis pathway. In the studies summarized in Chapter 2, we have characterized a pre-clinical in vivo model of PD involving aSyn overexpression in rat SN and used this model to investigate whether Nfe2L1 co-expression could alleviate aSyn neurotoxicity by reducing cytosolic aSyn levels via proteasome activation. Using unbiased stereology to determine nigral DA neuron cell counts, we found that Nfe2L1 may have a protective effect against aSyn-mediated nigral DA neurodegeneration. Surprisingly, we observed no increase in proteasome subunit expression through quantitative PCR or immunoblotting. However, by using a single-neuron analysis approach, we observed a significant increase in PSMC1 subunit expression, suggesting that Nfe2L1 expression could indeed lead to an upregulation of proteasome subunits and an increase in proteasome function. Future experiments will be aimed at determining whether Nfe2L1 expression results in an increase in proteasome activity, an enhancement of aSyn degradation, and a decrease in the burden of proteinase K-resistant (Lewy-like) aSyn aggregates in rat SN.
The ability to detect PD in the pre-symptomatic stage is necessary for the development of novel therapies to enable treatment prior to irreversible neuronal loss. Biomarkers with high sensitivity and specificity are critical for early PD detection. aSyn levels have been measured in human biofluids, such as blood and CSF, as a potential biomarker for PD diagnosis and for monitoring disease progression. aSyn can undergo a number of post-translational modifications (PTMs), and a particular form of the protein phosphorylated at serine 129 (pS129-aSyn) is enriched in Lewy bodies, making it an attractive candidate for biomarker studies. Although there are several antibodies targeting pS129, little is known about the influence of PTMs close to pS129 on the antibodies’ affinity for the pS129 epitope, or how these neighboring PTMs could affect assays developed to quantify pS129-aSyn in biofluids. In the studies summarized in Chapter 3, we characterized the impact of PTMs near pS129 on the affinity of currently available pS129-aSyn antibodies for their target antigen using biolayer interferometry (BLI). BLI analysis of the D1R1R pS129-aSyn antibody (Cell Signaling Technology) revealed that tyrosine phosphorylation or nitration at Y133 greatly reduced antibody affinity. In contrast, the MJF-R13 pS129-aSyn antibody (Abcam) was found to have reduced affinity for peptide targets nitrated or phosphorylated on Y125 or phosphorylated on Y133.
While aSyn is typically investigated as it relates to PD and other neurodegenerative disorders, recently reported evidence suggests that aSyn downregulation could be linked to an increased risk of neurodevelopmental disorders such as autism spectrum disorder (ASD). ASD is a complex neurodevelopmental disorder that presents with characteristic behavioral symptoms of social and communication impairments and restricted, repetitive behaviors. Diagnosis of ASD is based on the presentation of these behavioral symptoms, typically appearing at ~12 months of age, yet individuals don’t receive a diagnosis until the age of five. Early identification and early treatment are regarded as two of the most important factors for improving patient outcomes. SNCA gene deletions and loss-of-function duplications have been found in individuals with intellectual disability, developmental delay, and/or ASD, leading to the idea that reduced aSyn expression may be a biomarker for early ASD diagnosis and may play a role in ASD neuronal dysfunction. In the studies summarized in Chapter 4, we evaluated salivary aSyn as a potential biomarker for early ASD diagnosis and examined SNCA-/- iPSC-derived cortical neurons for indications of ASD-related neuronal anomalies. Our preliminary results suggest that salivary aSyn is reduced in individuals with neurodevelopmental disorders such as ASD and Fragile X Syndrome (FXS), a result consistent with findings of reduced aSyn in serum and plasma from ASD individuals. Furthermore, SNCA-/- iPSC-derived cortical neurons depleted of aSyn expression had increased soma size, a characteristic of iPSC-derived cortical neurons with ASD-associated mutations in the genes encoding MECP2 and TSC1/2 These results suggest that SNCA gene disruptions play a role in ASD-related neuronal anomalies and dysfunction.
Overall, the results presented in this thesis support a role for targeting aSyn protein expression in neurodegenerative and neurodevelopmental disorders, and they underscore the importance of designing aSyn biomarker immunoassays that faithfully report on each of these syndromes. Our data suggesting that Nfe2L1 could protect against nigral neurodegeneration by stimulating proteasome-mediated aSyn clearance imply that strategies to increase Nfe2L1-dependent transcriptional activity (e.g., using small molecule activators or gene therapy) could ameliorate pathological aspects of PD. The results presented here also highlight the need for sensitive and specific biomarker assays targeting multiple aSyn proteoforms, and they suggest that aSyn could be a viable biomarker for early ASD diagnosis. Finally, our findings provide the first evidence that aSyn down-regulation contributes to neuronal anomalies associated with ASD, in turn suggesting that strategies to increase cytosolic aSyn by preventing its degradation or through gene therapy could potentially mitigate ASD neuronal dysfunction.
- Doctor of Philosophy
- Medicinal Chemistry and Molecular Pharmacology
- West Lafayette