The excessive accumulation of alpha-synuclein (a normal protein expressed in nervous system) in the brain has been shown to contribute to the pathogenesis of Parkinson disease (PD). Thus, remedies directed at the reduction and clearance of alpha-synuclein represents a valid approach for treatment of PD. The objective of this proposal is to test novel and potent immunogens in a transgenic PD pre-clinical model, with the aim of developing active and passive vaccines targeting the clearance of alpha-synuclein for PD treatment.
Our specific aim is to generate and isolate a panel of conformational isomers of alpha-synuclein (designated as X-alphaSyn) for development as therapeutic vaccines for PD. X-alphaSyn isomers are highly aggregative and immunogenic. They have the potential to elicit high titer of antibodies that bind endogenous alpha-synuclein and thus decrease its concentration. We will conduct the following two studies: 1) To determine the immunogenicity of X-alphaSyn in human alpha-synuclein transgenic mice and to demonstrate that X-alphaSyn can raise high-titer antibodies that cross react with native alpha-synuclein; and 2) To evaluate the neuronal accumulation of alpha-synuclein aggregates and neurodegeneration in the immunized mice.
Relevance to Diagnosis/Treatment of Parkinson’s Disease: Successful completion of this proposal will provide effective and validated immunogens ready to enter preclinical studies to file an IND to conduct a Phase I clinical trial for PD treatment. We will achieve this by collaborating with companies with vaccine programs (e.g. IsoVax Therapeutics, Elan, Novartis). The success of this proposed study will also create the possibility to generate monoclonal antibodies for passive immunotherapy of PD.
X-alphaSyn isomers belong to a new class of protein isomers termed as Xisomers that have potential to be developed as active and passive therapeutic vaccines. Our current studies show that: 1) X-isomers exhibit increased immunogenicity as compared to the native protein; 2) X-isomers break immune tolerance and elicit high titers of anti-native protein antibodies. We anticipate that X-alphaSyn will exhibit similar immunological properties and will be promising candidates for the development of PD therapeutic vaccines.
Alpha-synuclein transgenic rodent models were immunized with three non-native alpha-synuclein isomers (X-Syn-a, X- Syn-b, and X-Syn-c) individually, and with native alpha-synuclein as a control immunogen. Results from the first assay showed that all three isomers displayed higher immune responses as compared with native alpha-synuclein, and that the antibodies generated cross reacted with native alpha-synuclein. Isomer X-Syn-b exhibited the highest immunogenicity and statistical significance. In addition, none of the immunized models exhibited weight loss or abnormal behaviors, suggesting the immunogens do not have overt toxicity. Ongoing work will continue to look at the time course of immunogenicity and the impact of immunization on alpha-synuclein pathological measures.
Homozygous alpha-synuclein transgenic pre-clinical models were immunized with three non-native alpha-synuclein isomers (X-alpha-synuclein-a, X alpha-synuclein-b, and X alpha-synuclein-c) and native alpha-synuclein was used as a control immunogen. None of the three X alpha-synucleins exhibited higher immunogenicity than native alpha-synuclein, which is contrary to what we expected. In fact, native alpha-synuclein exhibited the highest immunogenicity, which is consistent with the data published by Dr. Mashlia in 2005. There was variability in alpha-synuclein antibody levels among the immunized models in each group but the variability did not result from the measurement itself because every measurement was compared to the standard curve (spotted in the same plate) using pre-clinical model monoclonal anti-human alpha-synuclein antibody 4B12 (Abcam). In the X-alpha-synuclein-b immunized pre-clinical model no.182 and five native alpha-synuclein immunized mice, we observed both higher and consistent alpha-synuclein antibody levels and decreased brain alpha-synuclein levels, which strongly suggests that alpha-synuclein antibodies may produce a therapeutic effect. In addition, no significant pathology was detected by immunostaining with alpha-synuclein and synaptophysin antibodies. Finally, none of immunized models exhibited significant weight loss or abnormal behaviors, suggesting the immunogens did not have overt toxicity.