Introduction: In Parkinson’s disease (PD), the degeneration of dopaminergic neurons of the substantia nigra pars compacta results from the pathological accumulation of alpha-synuclein. Symptoms worsen as these neurons are lost despite dopamine replacement, creating a critical need to develop better neuroprotective therapies. Our approach utilizes gene therapy to introduce C3 transferase (C3), a bacterial exoenzyme that inhibits RhoA, a key intracellular inhibitory GTPase responsible for propagating axon growth collapse and cell death. We hypothesize that long-term endogenous expression of C3 in dopaminergic neurons will not exert major neurotoxic effects, allowing further assessment of its neuroprotective potential in PD.
Methods: We have developed floxed adeno-associated viral (AAV-DIO-Ef1a-GFP-2A-C3) vectors for gene delivery of C3 for long-term expression in the central nervous system (CNS) in a cell-autonomous (endogenous) fashion. These vectors were injected via a unilateral stereotactic injection into the substantia nigra of TH-Cre mice for specific expression in the nigrostriatal pathway. Mice were assessed for behavioral abnormalities by amphetamine-induced rotational analysis and compared to control mice receiving the viral vector without C3 and mice lesioned with 6-hydroxydopamine (6-OHDA). After 28 days, mice were sacrificed for brain sectioning and immunohistochemistry with markers of cell death.
Results: Mice injected with C3 exhibited similar ipsilateral rotations to the site of injection in comparison to control mice and significantly fewer ipsilateral rotations in comparison to mice lesioned with 6-OHDA. Upon sectioning and staining, C3 was expressed endogenously in dopaminergic neurons in a continuous manner with strong expression of tyrosine hydroxylase and weak expression of cleaved activated caspase 3, indicating cell viability and minimal induction of apoptosis.
Conclusions: These findings demonstrate that virally-transfected dopaminergic neurons express C3 without significant neurotoxicity, supporting its safety in long-term endogenous expression. Utilization of gene therapy with C3 should be investigated further as a therapeutic option for patients affected with PD.
Patient Care: Our findings support the safety of C3 gene therapy as a possible therapeutic option for the treatment of PD. C3 has shown potential in preventing axon growth collapse and cell death and offers promise for neuroprotection if delivered long-term via gene therapy. Assessing its safety is critical in future investigations of C3 gene therapy as a therapeutic option for patients affected with PD.
Learning Objectives: By conclusion of this session, participants should be able to:
1.) Describe the role of RhoA activation in promoting axon growth collapse and cell death and the ability of C3 transferase to inhibit Rho activation.
2.) Discuss the ability of C3 transferase gene therapy to inhibit RhoA activation with minimal long-term neurotoxicity to transfected neurons.
3.) Identify C3 transferase gene therapy for neuroprotection as a novel approach in the treatment of PD.
References: Dehay, Benjamin, et al. "Targeting a-synuclein for treatment of Parkinson's disease: mechanistic and therapeutic considerations." The Lancet Neurology 14.8 (2015): 855-866.
Gross, Robert E., et al. "The pivotal role of RhoA GTPase in the molecular signaling of axon growth inhibition after CNS injury and targeted therapeutic strategies." Cell transplantation 16.3 (2007): 245-262.
Gu, Haigang, et al. "Inhibition of the Rho signaling pathway improves neurite outgrowth and neuronal differentiation of mouse neural stem cells." International journal of physiology, pathophysiology and pharmacology 5.1 (2013): 11.
Gutekunst, Claire-Anne, et al. "C3 transferase gene therapy for continuous conditional RhoA inhibition." Neuroscience 339 (2016): 308-318.