Developing and characterizing cellular models for neurodegenerative diseases.

The focus of this work was to develop and validate in vitro cell culture models of neurodegenerative diseases, with a focus on Amyotrophic Lateral Sclerosis (ALS). ALS is a devastating neurodegenerative disease characterized by the progressive loss of motor neurons, eventually leading to paralysis and death. A hallmark of ALS, as well as other neurodegenerative diseases, is the accumulation of wild-type and mutant proteins into cytoplasmic aggregates. Autophagy is a homeostatic process required for the regulation and recycling of long-lived and misfolded proteins. As such, we focused on investigating the role of autophagy in ALS. Using ALS patient and control dermal fibroblasts, we began by investigating whether ALS-related proteins are regulated by the autophagy pathway, and whether patient cells would demonstrate any impairments of the pathway. Our primary findings demonstrated that ALS patient fibroblasts demonstrate both impaired autophagic initiation and impaired autophagic flux, that is, the turnover of targeted proteins. These cells are additionally more sensitive to oxidative stress induced apoptosis, possibly due to impaired autophagic mechanisms. We next investigated the role astrocytes may play in the autophagy pathway in the setting of ALS. For this, induced pluripotent stem cells were generated from three ALS patients and three healthy controls. These were characterized and differentiated to motor neurons and astrocytes, where we demonstrated that patient astrocytes can induce cytotoxicity in both healthy and control motor neurons, as previously reported. We hypothesized that this cytotoxicity may be mediated through impairments of the autophagy pathway. ALS patient and control iPSC derived astrocytes demonstrate similar levels of autophagy components. However, when we examined levels of mTOR, a protein involved in the regulation of many cellular processes, we show that it is greatly increased in patients relative to controls. We further show that conditioned medium from both ALS patient and control astrocytes induces autophagy in HEK293T cells via upregulation of pro-autophagic proteins BECLIN-1 and ATG12. Interestingly, LC3B, a protein required for the selective degradation of p62 and other cargo, is also upregulated, but only in response to control conditioned medium. Patient conditioned medium further induces an increase in p62 puncta accumulation in cells, indicating impaired autophagic flux. Whether increased mTOR expression is responsible for impaired activation of LC3B is unclear, but these data suggest the possibility that patient astrocytes may induce accumulation of autophagy and p62-selective proteins by dysregulating the balance between autophagosome formation and turnover. We additionally aimed to develop novel methods for the direct induction of neural stem cells from dermal fibroblasts. Building on work published by other groups, we investigated whether overexpression of IGF2, a growth factor highly upregulated in hippocampal neural stem cells, or RARΒ∆384, could improve reprogramming efficiency. We successfully generated and characterized neural stem cells using combinations of SOX2/BRN2/FOXG1, SOX2/BRN2/FOXG1/IGF2 and SOX2/BRN2/FOXG1/RARΒΔ384. RT-PCR analysis demonstrated that these populations retain a ventral hindbrain regional identity. These cells were capable of generating oligodendrocyte progenitor cells, mature astrocytes and glutamatergic and GABAergic neurons, indicating tri-lineage differentiation potential. However, they did not generate motor neurons or dopaminergic neurons. RARΒΔ384 increased the numbers of GABAergic neurons obtained at three weeks of differentiation, while IGF2 was shown to increase survival of GABAergic neurons over a 5 week period. Despite not showing propensity for motor neuron generation, these cells may be useful for the rapid generation of astrocytes for disease modeling in ALS, avoiding the need for the generation of iPSCs.

Rights

Attribution-NonCommercial-NoDerivs 3.0 Ireland

cbn

Collections

University of Galway Theses (PhD Theses)