Objective/Rationale:
We seek to develop small molecule drugs that promote the cellular process of autophagy as disease-modifying therapy for Parkinson's disease (PD). Autophagy is a highly conserved cellular process in which aggregate-prone proteins are targeted for cellular degradation. In Parkinson's disease aggregates of alpha-synuclein are a hallmark pathological feature. We hypothesize that promoting the autophagic clearance of alpha-synuclein will reduce PD-related pathology.
Project Description:
In vitro cellular models have been established and validated as a screening strategy for the identification of autophagy-promoting compounds. Preliminary data have been established to demonstrate that these same assays can be used to provide mechanistic insight into the cellular pathways being modified by the identified compounds. In addition, we have also established that compounds with autophagy-promoting activity can reduce the level of synuclein aggregation in a cellular model of synuclein-aggregation, and this assay will be used for further characterization of compounds of interest. Proof-of-concept studies will be conducted in vivo, in an animal model of synuclein aggregation, to demonstrate the reduction of aggregation, and such a model will be used to evaluate candidate lead compounds with good pharmacokinetic properties.
Relevance to Diagnosis/Treatment of Parkinson's Disease:
Multiple lines of independent experimental evidence support a role of autophagy in clearing aggregated alpha-synuclein both in vitro and in vivo. Both genetic and biochemical evidence links high levels of aggregated alpha-synuclein, the major component of Lewy body inclusions in brain regions vulnerable in PD, to sporadic and familial PD. The ability to clear aggregated alpha-synuclein represents a potential disease-modifying approach to PD.
Anticipated Outcome:
We will identify small molecule modulators of autophagy and establish their mechanism of action to allow an elaboration of compound structure/activity relationship and which cellular pathways are being modified. Based on an evaluation of the pharmacokinetic properties of candidate lead molecules, compounds will be evaluated for their ability to reduce alpha-synuclein aggregates in an animal model of synuclein aggregation. Such validation enables further evaluation and development of lead candidates as disease-modifying drugs for PD.
Final Outcome
The team has developed two new cellular assays to measure autophagy flux and have screened a small proprietary library, comprised of approximately 6,000 compounds, which are well characterized and target about 1,100 different mechanisms, ranging from receptors to enzymes. One hit of particular interest is a kinase inhibitor that stimulates autophagy flux in a novel way. We have developed and validated a number of cell-based assays to evaluate the effects of autophagy inducers on alpha-synuclein levels and the pathophysiological effects of this protein on autophagy flux and mitochondrial mobility, a novel and sensitive endpoint.
In order to test this hypothesis in vivo, we have characterized the alpha-synuclein A53T models to ascertain which are the most sensitive and relevant endpoints to evaluate. Our results demonstrate that these models may be useful to examine autophagy because they exhibit early and late deficits and develop alpha-synuclein pathology that increases with age, similar to Parkinson's disease patients. However, robust measures of autophagy flux in vivo still represent a significant gap in the field and are critical for:
1. Confirming compound mechanism of action in the relevant biological compartment
2. Potential biomarker to guide dose selection for therapeutic dosing
3. Developing PK/PD relationships for autophagy modulators
Presentations & Publications
Published manuscripts:
- W. Shen, A.G. Henry, K.L. Paumier, L. Li, K. Mou, J. Dunlop, Z. Berger, W.D. Hirst. (2014) Inhibition of glucosylceramide synthase enhances autophagy flux and decreases mutant α-synuclein levels in neurons.: J. Neurochem. 2014 Feb 4. doi: 10.1111/jnc.12672. [Epub ahead of print]
- L. Li, S. Nadanaciva, Z. Berger, K. Paumier, W. Shen, J. Schwartz, K. Mou, J. Dunlop, W.D. Hirst. Human A53T α-synuclein causes reversible deficits in mitochondrial function and dynamics in primary mouse cortical neurons. PLoS One 2013 Dec 31;8(12):e85815. doi: 10.1371/journal.pone.0085815. eCollection 2013.
- Paumier KL, Sukoff Rizzo SJ, Berger Z, Chen Y, Gonzales C, Kaftan E, Li L, Lotarski S, Monaghan M, Shen W, Stolyar P, Vasilyev D, Zaleska M, Hirst WD*, Dunlop J. (2013) Behavioral Characterization of A53T Mice Reveals Early and Late Stage Deficits Related to Parkinson's Disease. PLoS One. 2013 Aug 1;8(8):e70274. doi: 10.1371/journal.pone.0070274.
Submitted manuscript:
- Berger Z., Paumier P., Chen Y., Loos P., Shen W., Li L., Mou K., Samaroo H., Schwartz J., Dunlop J., Hirst W. D. (submitted PLosOne). Diverse effects of aggregate-prone proteins and potential autophagy enhancers on autophagy flux in primary neurons.
Abstracts/conference presentations:
- Berger Z. (2012). Drug Discovery Efforts in Autophagy - Therapeutic Targets for Alzheimer's and Parkinson's Diseases. New York Academy of Sciences, oral presentation, New York, USA.
- Berger Z., Shen W., Li L., Paumier K, Chen Y, Loos P., Mou K., Schwartz J., Gebhard D., Doyonnas R., Engle S., Dunlop J., Hirst W. D. (2012). Assessing autophagy flux in primary neurons and other cell based assays – evaluation of putative autophagy enhancers. Gordon Research Conference, Ventura, CA.
- Shen W., Hallowell S., Paumier K. L., Li L., Loos P., Gebhard D., Doyonnas R., Stepan A. F., Wager T. T., Dunlop J., Hirst W. D., Berger Z. (2012). Autophagy enhancers as potential therapeutics for neurodegenerative diseases: using a flow cytometry based assay to monitor autophagy flux. Society for Neuroscience Meeting, New Orleans, USA.
- Li L., Nadanaciva S., Berger Z., Paumier K., Shen W., Schwartz J., Mou K., Dunlop J., Hirst W.D. (2012), Parkinson’s disease-associated a-synuclein mutation impairs mitochondrial trafficking in neurons, Mitochondrial Trafficking and Function in Neuronal Health and Disease Abstract. Abcam conferences, Boston, MA.
- Gebhard D., Doyonnas R., Bradley J., Hallowell S., Shen W., Berger Z. (2012). High Throughput Flow Cytometry Assay for p62-GFP Autophagic Flux. CYTO2012, Leipzig, Germany.
- Paumier K., Gonzales C., Rizzo S., Stoylar P., Lotarski S., Hsu C., Roof R., Richter K. E. G, Berger Z., Li L., Monaghan M., Hirst W. D., Zaleska M. M., Dunlop J. (2012), Age-dependant phenotypic analysis of the A53T mouse model of Parkinson’s disease. Presented at the International Conference on Alzheimer's Drug Discovery
- Berger Z., Richter K., Li L., Paumier K., Atchison K., Engle S., Riddell D., Dunlop J., Hirst W. D. (2011). Evaluation of putative small molecule autophagy enhancers in primary neurons and neuronal cell lines. Society for Neuroscience Meeting, Washington D.C., USA.
- Li L., Richter K. E. G., Berger Z., Paumier K. P., Hirst W. D., Milici A. J., Dunlop J. (2011). Loperamide inhibits autophagic flux in H4 neuroglioma cells. Society for Neuroscience Meeting, Washington D.C., USA.
- Paumier K., Gonzales C., Stolyar P., Lotarski S., Hsu C., Richter K.E.G., Berger Z., Li L., Monaghan M., Hirst W.D., Zaleska M., Dunlop J (2011). Characterization of the phenotypic and age-dependant changes exhibited by the A53T mouse model of Parkinson’s disease. Society for Neuroscience Meeting, Washington D.C., USA.
April 2014