Recommended if funds allow
Statement of Benefit to California:
SYNOPSIS: This project aims to create an in vitro Parkinson's disease model using hESCS over-expressing PD pathogenic mutants of alpha synuclein and LRRK2. The hypothesis to be tested is that PD-associated genes function in hESC-derived DA neurons as in adult human brain PD DA neurons. Three specific aims are proposed that: 1. Generate hESC lines that will stably express wild type and PD pathogenic LRRK2 and wild type and PD pathogenic alpha synuclein; 2. Determine the effects of the synuclein and LRRK variants on hESCs and dopamine neurons derived from them; and, 3. Determine the effects of oxidative stress, known to be a factor in PD etiology from other in vitro and in vivo models, on these different normal and at-risk hESC lines and their DA-progeny. SIGNIFICANCE AND INNOVATION: There is no question that hESCs offer the possibility to model and study human disease in vitro (and potentially in vivo) by way of introducing gene mutations known to be involved in, e.g., familial neurodegenerative disease. The present study will look at six recently characterized genes known to be individually linked to early onset familial forms of PD. Establishing the alpha synuclein and LRRK2 variants of hESCs will be of value to the field, e.g. for screening drugs and factors that may inhibit aggregate formation and death (although we have no reason to believe that these structures and consequences will appear in the new lines to be generated here). The goal of generating a differentiated DA neuron that carries a mutant pathogenic gene is an innovative one, as is the plan to challenge the cell with varying stresses. STRENGTHS: Models of human neurodegenerative disease suffer from the neurons of interest being terminally differentiated; hESCs can be propagated and remain undifferentiated enough and long enough to introduce disease-associated genes and study the development of disease in culture (where bioassays and high throughput screening can be performed in controlled and highly reliable experiments). The proposed studies of determining the effects of oxidative stress on DA neurons derived from hESC, potentiating their susceptibility, are also timely and interesting. The choices of MPP+, paraquat, and rotenone are appropriate. The proposal is straightforward and logical. The planned experiments may lead to new important information, i.e., they are worth doing. The PI, an Assistant Professor at the Burnham Institute for Medical Research, is a productive investigator who has experience investigating a number of neurodegenerative diseases, including PD WEAKNESSES: With the approved and unapproved lines to be generated/studied (e.g. selecting “20 clones” for each viral infection, with the unfortunate issue of “…Expression level of transgenes will vary from clone to clone…”), wildtype and pathogenic genes and controls, in normal and oxidative stress settings, this is a very ambitious project that certainly will go beyond the proposed timeline. The cell heterogeneity issue raised in the pitfalls of Aim 2 is not dealt with properly. If the PI wants to investigate altered differentiation effects of mutant synuclein and LRRK2, even if they improve their DA neuron generation well beyond their current 30% of the neurons in the culture, there will still be enough heterogeneity to complicate interpretation in these (and Aim 3) studies. As pointed out in the Methods section for Aim 1, preliminary studies looking at synuclein and LRRK2 variants showed no effect on cell survival of SH-SY5Y cells - what about aggregate formation? Does the PI believe that oxidative stress of these cells would have generated an effect? There is little documented evidence of sufficient experience/expertise from this PI to work with hESCs. As the PI points out, these transgenes may not produce a cellular phenotype (e.g. aggregates) in the hESC lines. This would greatly reduce significance for these studies. It would have been valuable for the PI to have detailed how he would quantitate and compare aggregation in Aim II. As the PI notes, the fact that only 30% of the cells following differentiation are TH-positive DA neurons (and 30% GABAergic) will make the completion and interpretation of specific aims II and III challenging. How can the PI be certain that the 30% of GABAergic cells (and not the DA neurons) are not responsible for or affect the phenotypes observed in cells that have been transduced with the mutant gene? Dr. Yi Sun, an Assistant Professor in UCLA has 15% effort on this proposal, however, no details as to what she will do are provided and no letter from Dr. Sun is included. It would have been valuable for the PI to have noted if this was collaboration and why this interaction was important. DISCUSSION: This is a productive investigator, well-established in field of neurodegeneration who has successfully implemented similar strategies in the past. Two important issues were raised: the application does not address the cellular heterogeneity in the cultures and how this will be dealt with; and it is not clear what the phenotype will be or if there will be one. The proposed readout of the experiments (formation of Lewey bodies) may be too restrictive. PROGRAMMATIC DISCUSSION: The Working Group voted to recommend to the ICOC that this application receive special consideration for funding if additional funds become available based on: 1)scientific merit of application - a very clear cut recommendation for funding and 2) this is research important to Parkinson's disease.